Please wait a minute...
Journal of Integrative Agriculture  2015, Vol. 14 Issue (5): 926-938    DOI: 10.1016/S2095-3119(14)60866-2
Animal Science · Veterinary Science Advanced Online Publication | Current Issue | Archive | Adv Search |
RNA-Seq transcriptome analysis of porcine cloned and in vitro fertilized blastocysts
 XU Wei-hua, LI Zi-cong, OUYANG Zhi-ping, YU Bo, SHI Jun-song, LIU De-wu, WU Zhen-fang
1、National Engineering Research Center for Breeding Swine Industry, South China Agricultural University, Guangzhou 510642,P.R.China
2、College of Life Science, Longyan University, Longyan 364012, P.R.China
3、Wen’s Research Institute, Yunfu 527439, P.R.China
Download:  PDF in ScienceDirect  
Export:  BibTeX | EndNote (RIS)      
摘要  Somatic nuclear transfer technology has become increasingly promising in biomedicine and agriculture. Whereas the approach remains inefficient and underlying mechanisms remain ambiguous. Although cloned embryos have similar in vitro developmental capacity as in vitro fertilized (IVF) embryos before implantation, they appeared to have much lower full-term developmental efficiency in pig and cattle, and thus it would be reasonable to postulate that profound distinction at the molecular level should exist between them. Herein, RNA sequencing technique was used to screen differentially expressed genes in cloned and IVF blastocysts, and in total 628 differentially expressed transcripts were obtained, among which, 280 transcripts are up-regulated and 348 transcripts are down-regulated in cloned blastocysts. Moreover, one statistically significant pathway associated with endoplasmic reticulum (ER) protein processing was enriched, and some ER-stress markers such as ATF4, ATF6, PDIA3, HSPA1B, HSP40 and HSP90 between cloned and IVF blastocysts were suggested. Additionally, some developmentally important genes such as lipid metabolism related genes (MGLL, DDHD2 and FADS2) and epigenetic modification genes (DNMT1, KDM5C and MBD3L5) were found differentially expressed between cloned and IVF embryos.

Abstract  Somatic nuclear transfer technology has become increasingly promising in biomedicine and agriculture. Whereas the approach remains inefficient and underlying mechanisms remain ambiguous. Although cloned embryos have similar in vitro developmental capacity as in vitro fertilized (IVF) embryos before implantation, they appeared to have much lower full-term developmental efficiency in pig and cattle, and thus it would be reasonable to postulate that profound distinction at the molecular level should exist between them. Herein, RNA sequencing technique was used to screen differentially expressed genes in cloned and IVF blastocysts, and in total 628 differentially expressed transcripts were obtained, among which, 280 transcripts are up-regulated and 348 transcripts are down-regulated in cloned blastocysts. Moreover, one statistically significant pathway associated with endoplasmic reticulum (ER) protein processing was enriched, and some ER-stress markers such as ATF4, ATF6, PDIA3, HSPA1B, HSP40 and HSP90 between cloned and IVF blastocysts were suggested. Additionally, some developmentally important genes such as lipid metabolism related genes (MGLL, DDHD2 and FADS2) and epigenetic modification genes (DNMT1, KDM5C and MBD3L5) were found differentially expressed between cloned and IVF embryos.
Keywords:  porcine       transcriptome       blastocyst       somatic cell nuclear transfer       in vitro fertilization  
Received: 03 April 2014   Accepted:
Fund: 

This study was supported by a grant from the National study was supported by a grant from the National High-Technology Research and Development Program of China (2011AA100304), and two grants from Guangdong Provincial Department of Science and Technology, China (2011A090700016 and 2011A020102003).

Corresponding Authors:  WU Zhen-fang, Tel/Fax: +86-20-85280369,E-mail: wzfemail@163.com     E-mail:  wzfemail@163.com
About author:  XU Wei-hua, Mobile: 13859594289, E-mail: xwh131@163.com;* These authors contributed equally to this work

Cite this article: 

XU Wei-hua, LI Zi-cong, OUYANG Zhi-ping, YU Bo, SHI Jun-song, LIU De-wu, WU Zhen-fang. 2015. RNA-Seq transcriptome analysis of porcine cloned and in vitro fertilized blastocysts. Journal of Integrative Agriculture, 14(5): 926-938.

Ahner A, Brodsky J L. 2004. Checkpoints in ER-associateddegradation: excuse me, which way to the proteasome?Trends in Cell Biology, 14, 474-478

Audic S, Claverie J M 1997. The significance of digital geneexpression profiles. Genome Research, 7, 986-995

Bauer B K, Isom S C, Spate L D, Whitworth K M, Spollen W G,Blake S M, Springer G K, Murphy C N, Prather R S 2010.Transcriptional profiling by deep sequencing identifiesdifferences in mRNA transcript abundance in in vivo-derivedversus in vitro-cultured porcine blastocyst stage embryos.Biology of Reproduction, 83, 791-798

Bendixen E, Danielsen M, Larsen K, Bendixen C. 2010.Advances in porcine genomics and proteomics-A toolboxfor developing the pig as a model organism for molecularbiomedical research. Briefings in Functional Genomics, 9,208-219

Bermejo A P, Lonergan P, Rath D, Gutiérrez A A, Rizos D. 2010.Developmental kinetics and gene expression in male andfemale bovine embryos produced in vitro with sex-sortedspermatozoa. Reproduction, Fertility and Development,22, 426-436

Bernardini C, Fantinati P, Zannoni A, Forni M, Tamanini C,Bacci M L. 2004. Expression of HSP70/HSC70 in swineblastocysts: effects of oxidative and thermal stress.Molecular Reproduction and Development, 69, 303-307

Betthauser J, Forsberg E, Augenstein M, Childs L, EilertsenK, Enos J, Forsythe T, Golueke P, Jurgella G, KoppangR, Lesmeister T, Mallon K, Mell G, Misica P, Pace M,Pfister-Genskow M, Strelchenko N, Voelker G, Watt S,Thompson S, et al. 2000. Production of cloned pigs fromin vitro systems. Nature Biotechnology, 18, 1055-1059

Brodsky J L, Wojcikiewicz R J. 2009. Substrate-specificmediators of ER associated degradation (ERAD). CurrentOpinion Cell Biology, 21, 516-521

Burton G J, Yung H W, Cindrova-Davies T, Charnock-JonesD S. 2009. Placental endoplasmic reticulum stress andoxidative stress in the pathophysiology of unexplainedintrauterine growth restriction and early onset preeclampsia.Placenta, 30(Suppl A), S43-48

Canepa M J, Mucci N, Kaiser G, Mutto A. 2011. Stress indicatorgenes expression pattern in bovine blastocysts productionby in vitro fertilization and somatic cell nuclear transfer.Reproduction, Fertility and Development, 24, 159.Chae J I, Lee K S, Kim D J, Han Y M, Lee D S, Lee K K, KooD B. 2009. Abnormal gene expression in extraembryonic tissue from cloned porcine embryos. Theriogenology, 71,323-333

Chan M M, Smith Z D, Egli D, Regev A, Meissner A. 2012.Mouse ooplasm confers context-specific reprogrammingcapacity. Nature Genetics, 44, 978-980

Ciccone D N, Su H, Hevi S, Gay F, Lei H, Bajko J, Xu G, LiE, Chen T. 2009. KDM1B is a histone H3K4 demethylaserequired to establish maternal genomic imprints. Nature,461, 415-418

Esfandiari N, Falcone T, Goldberg J M, Agarwal A, Sharma RK. 2007. Heat-shock proteins modulate the incidence ofapoptosis and oxidative stress in preimplantation mouseembryos. Fertility and Sterility, 87, 1214-1217

Feng C, Chen M, Xu C J, Bai L, Yin X R, Li X, Allan A C,Ferguson I B, Chen K S. 2012. Transcriptomic analysisof Chinese bayberry (Myrica rubra) fruit development andripening using RNA-Seq. BMC Genomics, 13, 19.

Gil M A, Cuello C, Parrilla I, Vazquez J M, Roca J, MartinezE A. 2010. Advances in swine in vitro embryo productiontechnologies. Reproduction in Domestic Animals, 45(Suppl.2), 40-48

Howell C Y, Bestor T H, Ding F, Latham K E, Mertineit C, TraslerJ M, Chaillet J R. 2001. Genomic imprinting disrupted bya maternal effect mutation in the Dnmt1 gene. Cell, 104,829-838

Inoue K, Kohda T, Sugimoto M, Sado T, Ogonuki N, MatobaS, Shiura H, Ikeda R, Mochida K, Fujii T, Sawai K, OtteA P, Tian X C, Yang X , Ishino F, Abe K, Ogura A. 2010.Impeding Xist expression from the active X chromosomeimproves mouse somatic cell nuclear transfer. Science,330, 496-499

Isom S C, Stevens J R, Li R, Spollen W G, Cox L, Spate L D,Murphy C N, Prather R S. 2013. Transcriptional profilingby RNA-Seq of peri-attachment porcine embryos generatedby a variety of assisted reproductive technologies.Physiological Genomics, 45, 577-589

Iwase S, Lan F, Bayliss P, de la Torre-Ubieta L, Huarte M, QiH H, Whetstine J R, Bonni A, Roberts T M, Shi Y. 2007.The X-linked mental retardation gene SMCX/JARID1Cdefines a family of histone H3 lysine 4 demethylases. Cell,128, 1077-1088

Jiang L, Lai L, Samuel M, Prather R S, Yang X, Tian X C. 2008.Expression of X-linked genes in deceased neonates andsurviving cloned female piglets. Molecular Reproductionand Development, 75, 265-273

Kurome M, Geistlinger L, Kessler B, Zakhartchenko V, KlymiukN, Wuensch A, Richter A, Baehr A, Kraehe K, BurkhardtK, Flisikowski K, Flisikowska T, Merkl C, Landmann M,Durkovic M, Tschukes A, Kraner S, Schindelhauer D, PetriT, Kind A, et al. 2013. Factors influencing the efficiencyof generating genetically engineered pigs by nucleartransfer: multi-factorial analysis of a large data set. BMCBiotechnology, 13, 43.

Lian I A, Løset M, Mundal S B, Fenstad M H, Johnson M P, EideI P, Bjørge L, Freed K A, Moses E K, Austgulen R. 2011.Increased endoplasmic reticulum stress in decidual tissuefrom pregnancies complicated by fetal growth restrictionwith and without pre-eclampsia. Placenta, 32, 823-829

Li R, Yu C, Li Y, Lam T W, Yiu S M, Kristiansen K, Wang J.2009. SOAP2: An improved ultrafast tool for short readalignment. Bioinformatics, 25, 1966-1967

Li Z, Shi J, Liu D, Zhou R, Zeng H, Zhou X, Mai R, ZengS, Luo L, Yu W, Zhang S, Wu Z. 2013. Effects of donorfibroblast cell type and transferred cloned embryo numberon the efficiency of pig cloning. Cellular Reprogramming,15, 35-42

Macháty Z, Day B N, Prather R S. 1998. Development ofearly porcine embryos in vitro and in vivo. Biology ofReproduction, 59, 451-455

Matoba S, Inoue K, Kohda T, Sugimoto M, Mizutani E, OgonukiN, Nakamura T, Abe K, Nakano T, Ishino F, Ogura A. 2011.RNAi-mediated knockdown of Xist can rescue the impairedpostimplantation development of cloned mouse embryos.Proceedings of the National Academy of Sciences of theUnited States of America, 108, 20621-20626

McKeegan P J, Sturmey R G. 2011. The role of fatty acidsin oocyte and early embryo development. Reproduction,Fertility and Development, 24, 59-67

Mortazavi A, Williamsm B A, McCue K, Schaeffer L, Wold B.2008. Mapping and quantifying mammalian transcriptomesby RNA-Seq. Nature Methods, 5, 621-628

Onishi A, Iwamoto M, Akita T, Mikawa S, Takeda K, Awata T,Hanada H, Perry A C. 2000. Pig cloning by microinjectionof fetal fibroblast nuclei. Science, 289, 1188-1190

Ooi S K, Qiu C, Bernstein E, Li K, Jia D, Yang Z, Erdjument-Bromage H, Tempst P, Lin S P, Allis C D, Cheng X, BestorT H. 2007. DNMT3L connects unmethylated lysine 4 ofhistone H3 to de novo methylation of DNA. Nature, 448,714-717

Peat J R, Reik W. 2012. Incomplete methylation reprogrammingin SCNT embryos. Nature Genetics, 44, 965-966

Pfister-Genskow M, Strelchenko N, Voelker G, Watt S,Thompson S, Bishop M. 2000. Production of cloned pigsfrom in vitro systems. Nature Biotechnology, 18, 1055-1059

Polejaeva I A, Chen S H, Vaught T D, Page R L, Mullins J, Ball S,Dai Y, Boone J, Walker S, Ayares D L, Colman A, CampbellK H. 2000. Cloned pigs produced by nuclear transfer fromadult somatic cells. Nature, 407, 86-90

Prather R S, Hawley R J, Carter D B, Lai L, Greenstein J L.2003. Transgenic swine for biomedicine and agriculture.Theriogenology, 59, 115-123

Somfai T, Ozawa M, Noguchi J, Kaneko H, Karja N W, FahrudinM, Nakai M, Maedomari N, Dinnyés A, Nagai T, Kikuchi K.2008. In vitro development of polyspermic porcine oocytes:Relationship between early fragmentation and excessivenumber of penetrating spermatozoa. Animal ReproductionScience, 107, 131-147

Song B S, Kim J S, Yoon S B, Lee K S, Koo D B, Lee D S,Choo Y K, Huh J W, Lee S R, Kim S U, Kim S H, Kim H M,Chang K T. 2011. Inactivated Sendai-virus-mediated fusionimproves early development of cloned bovine embryosby avoiding endoplasmic-reticulum-stress-associated apoptosis. Reproduction, Fertility and Development, 23,826-836

Sturmey R G, Reis A, Leese H J, McEvoy T G. 2009. Role offatty acids in energy provision during oocyte maturationand early embryo development. Reproduction in DomesticAnimals, 44(Suppl 3), 50-58

Szegezdi E, Logue S E, Gorman A M, Samali A. 2006. Mediatorsof endoplasmic reticulum stress-induced apoptosis. EMBOReports, 7, 880-885

Travers K J, Patil C K, Wodicka L, Lockhart D J, WeissmanJ S, Walter P. 2000. Functional and genomic analysesreveal an essential coordination between the unfoldedprotein response and ER-associated degradation. Cell,101, 249-258

Van M G, Voelker D R, Feigenson G W. 2008. Membrane lipids:where they are and how they behave. Nature ReviewsMolecular Cell Biology, 9, 112-124

Whitworth K M, Agca C, Kim J G, Patel R V, Springer G K,Bivens N J, Forrester L J, Mathialagan N, Green J A, PratherR S. 2005. Transcriptional profiling of pig embryogenesisby using a 15-K member unigene set specific for pigreproductive tissues and embryos. Biology of Reproduction,72, 1437-1451

Whitworth K M, Prather R S. 2010. Somatic cell nucleartransfer efficiency: how can it be improved through nuclearremodeling and reprogramming? Molecular Reproductionand Development, 77, 1001-1015

Whitworth K M, Spate L D, Li R, Rieke A, Sutovsky P, GreenJ A, Prather R S. 2010. Activation method does not alterabnormal placental gene expression and development incloned pigs. Molecular Reproduction and Development,77, 1016-1030

Whyte J J, Prather R S. 2011. Genetic modifications of pigsfor medicine and agriculture. Molecular Reproduction andDevelopment, 78, 879-891

Wu L L, Russell D L, Norman R J, Robker R L. 2012.Endoplasmic reticulum (ER) stress in cumulus-oocytecomplexes impairs pentraxin-3 secretion, mitochondrialmembrane potential (ΔΨm), and embryo development.Molecular Endocrinology, 26, 562-573

Xu W, Li Z, Yu B, He X, Shi J, Zhou R, Liu D, Wu Z. 2013.Effects of DNMT1 and HDAC inhibitors on gene-specificmethylation reprogramming during porcine somatic cellnuclear transfer. PLOS ONE, 8, e64705.

Yang X, Smith S L, Tian X C, Lewin H A, Renard J P, WakayamaT. 2007. Nuclear reprogramming of cloned embryos andits implications for therapeutic cloning. Nature Genetics,39, 295-302

Yang A S, Estécio M R, Doshi K, Kondo Y, Tajara E H, IssaJ P. 2004. A simple method for estimating global DNAmethylation using bisulfite PCR of repetitive DNA elements.Nucleic Acids Research, 32, e38.

Yoshioka K, Suzuki C, Itoh S, Kikuchi K, Iwamura S, RodriguezM H. 2003. Production of piglets derived from in vitroproducedblastocysts fertilized and cultured in chemicallydefined media: effects of theophylline, adenosine, andcysteine during in vitro fertilization. Biology of Reproduction,69, 2092-2099

Zhang J Y, Diao Y F, Oqani R K, Han R X, Jin D I. 2012.Effect of endoplasmic reticulum stress on porcine oocytematuration and parthenogenetic embryonic developmentin vitro. Biology of Reproduction, 86, 128.
[1] Meixue Sun, Tong Li, Yingjie Liu, Kenneth Wilson, Xingyu Chen, Robert I. Graham, Xianming Yang, Guangwei Ren, Pengjun Xu. A dicistrovirus increases pupal mortality in Spodoptera frugiperda by suppressing protease activity and inhibiting larval diet consumption[J]. >Journal of Integrative Agriculture, 2024, 23(8): 2723-2734.
[2] Qian Wang, Huimin Cao, Jingcheng Wang, Zirong Gu, Qiuyun Lin, Zeyan Zhang, Xueying Zhao, Wei Gao, Huijun Zhu, Hubin Yan, Jianjun Yan, Qingting Hao, Yaowen Zhang. Fine-mapping and primary analysis of candidate genes associated with seed coat color in mung bean (Vigna radiata L.)[J]. >Journal of Integrative Agriculture, 2024, 23(8): 2571-2588.
[3] Jiaxing Wei, Hong Yan, Jie Ren, Guangyue Li, Bo Zhang, Xuenong Xu.

Acaricidal effect of the antimicrobial metabolite xenocoumacin 1 on spider mite control [J]. >Journal of Integrative Agriculture, 2024, 23(3): 948-959.

[4] Qi Gao, Yongzhi Feng, Ting Gong, Dongdong Wu, Xiaoyu Zheng, Yizhuo Luo, Yunlong Yang, Zebu Song, Lang Gong, Guihong Zhang.

Porcine enteric alphacoronavirus infection increases lipid droplet accumulation to facilitate the virus replication [J]. >Journal of Integrative Agriculture, 2024, 23(3): 988-1005.

[5] Liyao Su, Min Wu, Tian Zhang, Yan Zhong, Zongming (Max) Cheng.

Identification of key genes regulating the synthesis of quercetin derivatives in Rosa roxburghii through integrated transcriptomics and metabolomics [J]. >Journal of Integrative Agriculture, 2024, 23(3): 876-887.

[6] XIAO Ning, LÜ Yun-yun, LI Jian-nan, CHEN Chang-feng, LIN Hui-xing, FAN Hong-jie. Preparation and application of a novel monoclonal antibody specific for the heat shock protein 60 of Lawsonia intracellularis[J]. >Journal of Integrative Agriculture, 2023, 22(9): 2824-2833.
[7] XU Kui, ZHOU Yan-rong, SHANG Hai-tao, XU Chang-jiang, TAO Ran, HAO Wan-jun, LIU Sha-sha, MU Yu-lian, XIAO Shao-bo, LI Kui. Pig macrophages with site-specific edited CD163 decrease the susceptibility to infection with porcine reproductive and respiratory syndrome virus[J]. >Journal of Integrative Agriculture, 2023, 22(7): 2188-2199.
[8] SHAN Yan-ju, JI Gai-ge, ZHANG Ming, LIU Yi-fan, TU Yun-jie, JU Xiao-jun, SHU Jing-ting, ZOU Jian-min. Use of transcriptome sequencing to explore the effect of CSRP3 on chicken myoblasts[J]. >Journal of Integrative Agriculture, 2023, 22(4): 1159-1171.
[9] SONG Xiang-peng, XIA Ying-ju, XU Lu, ZHAO Jun-jie, WANG Zhen, ZHAO Qi-zu, LIU Ye-bing, ZHANG Qian-yi, WANG Qin. A multiplex real-time PCR assay for simultaneous detection of classical swine fever virus, African swine fever virus and atypical porcine pestivirus[J]. >Journal of Integrative Agriculture, 2023, 22(2): 559-567.
[10] ZHAO Wen-juan, YUAN Xiao-ya, XIANG Hai, MA Zheng, CUI Huan-xian, LI Hua, ZHAO Gui-ping. Transcriptome-based analysis of key genes and pathways affecting the linoleic acid content in chickens[J]. >Journal of Integrative Agriculture, 2023, 22(12): 3744-3754.
[11] LIAO Guang-lian, HUANG Chun-hui, JIA Dong-feng, ZHONG Min, TAO Jun-jie, QU Xue-yan, XU Xiao-biao. A high-quality genome of Actinidia eriantha provides new insight into ascorbic acid regulation[J]. >Journal of Integrative Agriculture, 2023, 22(11): 3244-3255.
[12] ZHANG Rui-xing, ZHANG Ni-nan, WANG Ya-xiu, Khan ABID, MA Shuai, BAI Xue, ZENG Qi, PAN Qi-ming, LI Bao-hua, ZHANG Lu-gang. Blue light induces leaf color change by modulating carotenoid metabolites in orange-head Chinese cabbage (Brassica rapa L. ssp. pekinensis)[J]. >Journal of Integrative Agriculture, 2023, 22(11): 3296-3311.
[13] SU He-nan, YUAN Yu-xiang, YANG Shuang-juan, WEI Xiao-chun, ZHAO Yan-yan, WANG Zhi-yong, QIN Liu-yue, YANG Zhi-yuan, NIU Liu-jing, LI Lin, ZHANG Xiao-wei. Comprehensive analysis of the full-length transcripts and alternative splicing involved in clubroot resistance in Chinese cabbage[J]. >Journal of Integrative Agriculture, 2023, 22(11): 3284-3295.
[14] WANG Xiao-dong, CAI Ying, PANG Cheng-ke, ZHAO Xiao-zhen, SHI Rui, LIU Hong-fang, CHEN Feng, ZHANG Wei, FU San-xiong, HU Mao-long, HUA Wei, ZHENG Ming, ZHANG Jie-fu. BnaSD.C3 is a novel major quantitative trait locus affecting semi-dwarf architecture in Brassica napus L.[J]. >Journal of Integrative Agriculture, 2023, 22(10): 2981-2992.
[15] ZHENG Qi, HU Rong-cui, ZHU Cui-yun, JING Jing, LOU Meng-yu, ZHANG Si-huan, LI Shuang, CAO Hong-guo, ZHANG Xiao-rong, LING Ying-hui. Identification of transition factors in myotube formation from proteome and transcriptome analyses[J]. >Journal of Integrative Agriculture, 2023, 22(10): 3135-3147.
No Suggested Reading articles found!