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

Abstract

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

CLC Number:

S828.2

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.

Figures/Tables 3

References 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]	SUN Yawen, CHEN Siying, LI Kang, LENG Xuan, WANG Dong, PANG Yunwei. Strategies for Alleviating Cryoinjury of Porcine Vitrified-Oocytes [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(1): 36-44.
[2]	ZHOU Xianshan, HUANG Shihui, NIU Xi, RAN Xueqin, WANG Jiafu. Differential Expression Study of Structural Variation in the Ubiquitin Ligase 2 Gene of Xiang Pigs with Wrinkled Skin [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(1): 136-146.
[3]	WU Pingxian, WANG Junge, DIAO Shuqi, CHAI Jie, ZHA Lin, GUO Zongyi, CHEN Hongyue, LONG Xi. Analysis of Genetic Architecture Characteristics and Selection Signature by Imputed Whole Genome Sequencing Data in Rongchang Pigs [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(1): 147-158.
[4]	YANG Wenpan, LIU Xiangjie, LUO Dongxiang, CHEN Menghui, XIE Ying, FANG Yuexin, LIN Tingyan, LI Aimin, LI Wenjing, DENG Zheng, DING Nengshui. Research on Genomic Selection of Reproductive Traits in Landrace Pigs Based on Chip Data [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(1): 213-221.
[5]	FAN Dingkun, ZHANG Tao, JIAO Shuai, LU Wei, FU Yuze, YANG Hong, TU Yan, SHI Lingyuan, ZHANG Naifeng. Evaluation of Relative Bioavailability of Tricalcium Phosphate Produced by High-temperature Sintering Method in Weaned Piglets Based on Slope Ratio Method [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(1): 269-280.
[6]	WANG Xingyue, YANG Zhiyuan, LIN Jian, ZHANG Zixuan, ZHOU Yuting, CHENG Huimin, LIU Yuehuan, HU Ge. Development and Application of a Real-time Fluorescence Quantitative PCR Detection Method for Pigeon Circovirus [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(1): 335-342.
[7]	Jingjing TIAN, Xiaoqing WANG, Mianyan LI, Hailing WANG, Qitian WU, Lixian WANG, Longchao ZHANG, Fuping ZHAO. Analysis of the Whole Genome Run of Homozygosity (ROH) and Selection Signal in Beijing Black Pigs [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(9): 3833-3842.
[8]	Dong CHEN, Wenxuan ZHOU, Zhenjian ZHAO, Qi SHEN, Yang YU, Shengdi CUI, Junge WANG, Ziyang CHEN, Shixin YU, Jiamiao CHEN, Xiangfeng WANG, Pingxian WU, Zongyi GUO, Jinyong WANG, Guoqing TANG. Development of a Pig Intramuscular Fat Content and Eye Muscle Area Measurement System Based on Computer Vision Technology [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(9): 3843-3852.
[9]	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.
[10]	Cong REN, Hu ZHANG, Yuming WANG, Jingjing XIE, Renna SA, Feng ZHAO. Study on the Accuracy and Additivity of Effective Energy in Feed for Growing Pigs Predicted by Simulated Digestion Method [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(9): 3988-4000.
[11]	Shan ZHANG, Dahu LIU, Baojing LIU, Lin LIANG, Ruiying LIANG, Xinming TANG, Xusheng QIU, Chan DING, Jiabo DING, Shaohua HOU. Isolation, Identification and Pathogenicity Analysis of a Pigeon Paramyxovirus-1 Strain [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(9): 4051-4060.
[12]	Xiaojuan LIANG, Yushuang LI, Zhou FU, Duo TANG, Yingying LI, Shouwei WANG. Isolation, Culture and Adipogenic Differentiation of Pigeon Preadipocytes [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(8): 3482-3492.
[13]	Jing CHEN, Xuebei WU, Dongzhi MIAO, Chi ZHANG, Zhenyu GUO, Ying WANG. Comparative Analysis of Transcriptome of Pigeon Follicles at Early Stage of Laying Interval Reveals Genes Related to Follicular Development [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(8): 3503-3515.
[14]	Yue LI, Changchun ZHANG, Guangyu LIU, Mengyuan GAO, Chaojun FU, Jiabao XING, Sijia XU, Qiyuan KUANG, Jing LIU, Xiaopeng GAO, Heng WANG, Lang GONG, Guihong ZHANG, Yankuo SUN. Application and Analysis of Meta-transcriptomics Sequencing Technology in the Diagnosis of Viral Diarrhea Diseases in Piglets [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(8): 3579-3589.
[15]	Tengfei DOU, Jiahao WU, Ziyi WU, Liyao BAI, Xinjian LI, Xuelei HAN, Ruimin QIAO, Kejun WANG, Feng YANG, Yining WANG, Xiuling LI. Application Progress of Genomic Selection and Mating Allocation Techniques in Pig Breeding [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(7): 2795-2808.

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

RichHTML

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 3

References 40

Related Articles 15

Recommended Articles

Metrics

Comments