FREE SHIPPING over $40
OVER 9000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
Home / Equine Research Bank / Epigenetics Chromatin / Dynamics of 5-methylcytosine and 5-hydroxymethylcytosine dur...
Epigenetics & chromatin2017; 10; 13; doi: 10.1186/s13072-017-0120-x

Dynamics of 5-methylcytosine and 5-hydroxymethylcytosine during pronuclear development in equine zygotes produced by ICSI.

Abstract: Global epigenetic reprogramming is considered to be essential during embryo development to establish totipotency. In the classic model first described in the mouse, the genome-wide DNA demethylation is asymmetric between the paternal and the maternal genome. The paternal genome undergoes ten-eleven translocation (TET)-mediated active DNA demethylation, which is completed before the end of the first cell cycle. Since TET enzymes oxidize 5-methylcytosine to 5-hydroxymethylcytosine, the latter is postulated to be an intermediate stage toward DNA demethylation. The maternal genome, on the other hand, is protected from active demethylation and undergoes replication-dependent DNA demethylation. However, several species do not show the asymmetric DNA demethylation process described in this classic model, since 5-methylcytosine and 5-hydroxymethylcytosine are present during the first cell cycle in both parental genomes. In this study, global changes in the levels of 5-methylcytosine and 5-hydroxymethylcytosine throughout pronuclear development in equine zygotes produced in vitro were assessed using immunofluorescent staining. We were able to show that 5-methylcytosine and 5-hydroxymethylcytosine both were explicitly present throughout pronuclear development, with similar intensity levels in both parental genomes, in equine zygotes produced by ICSI. The localization patterns of 5-methylcytosine and 5-hydroxymethylcytosine, however, were different, with 5-hydroxymethylcytosine homogeneously distributed in the DNA, while 5-methylcytosine tended to be clustered in certain regions. Fluorescence quantification showed increased 5-methylcytosine levels in the maternal genome from PN1 to PN2, while no differences were found in PN3 and PN4. No differences were observed in the paternal genome. Normalized levels of 5-hydroxymethylcytosine were preserved throughout all pronuclear stages in both parental genomes. In conclusion, the horse does not seem to follow the classic model of asymmetric demethylation as no evidence of global DNA demethylation of the paternal pronucleus during the first cell cycle was demonstrated. Instead, both parental genomes displayed sustained and similar levels of methylation and hydroxymethylation throughout pronuclear development.
Get Full Text
Publication Date: 2017-03-15 PubMed ID: 28331549PubMed Central: PMC5353960DOI: 10.1186/s13072-017-0120-xGoogle Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
LinkedInCopy
  • Journal Article
  • Research Support
  • Non-U.S. Gov't

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research study explores how the levels of 5-methylcytosine and 5-hydroxymethylcytosine, involved in DNA methylation, change during the development of horse zygotes. The findings show that unlike in many other species, both compounds are consistently present in both paternal and maternal genomes, suggesting that horses do not follow the traditional model of genome-wide DNA demethylation.

Understanding the Concept: DNA Methylation and Its Role

  • Global epigenetic reprogramming, which includes the process of DNA methylation, is crucial for embryo development to establish totipotency, or the ability of an individual cell to divide and produce all the differentiated cells in an organism.
  • A classical model, first observed in mice, explains the process of genome-wide DNA demethylation as being unequal between the maternal and paternal genomes. The paternal genome undergoes active DNA demethylation mediated by TET (ten-eleven translocation) enzymes. This process is completed before the end of the first cell cycle.
  • TET enzymes have the role of catalyzing the oxidation of 5-methylcytosine to 5-hydroxymethylcytosine. The molecule 5-hydroxymethylcytosine is thought to be an intermediary stage on the path to DNA demethylation.
  • Contrastingly, the maternal genome is protected from active demethylation and undergoes DNA demethylation depending on replication.

The Unconventional Case: Horse Zygotes

  • Many species do not follow the classic model of asymmetric DNA demethylation. The levels of 5-methylcytosine and 5-hydroxymethylcytosine are maintained during the first cell cycle in both parental genomes.
  • The research study conducted focused on monitoring how the quantities of these compounds changed during the development of horse zygotes created in vitro.
  • It was observed that both 5-methylcytosine and 5-hydroxymethylcytosine were consistently present throughout the development with similar levels in both parental genomes. The patterns of Localization of these compounds, however, differed. While 5-hydroxymethylcytosine was evenly distributed in the DNA, 5-methylcytosine was found to be concentrated in specific regions.

Interpretation & Conclusions

  • Quantification of fluorescence revealed increased 5-methylcytosine levels in the maternal genome from PN1 to PN2; however, no differences were found in PN3 and PN4. No alterations were noticed in the paternal genome.
  • The normalized levels of 5-hydroxymethylcytosine were maintained throughout all pronuclear stages in both parental genomes.
  • Based on these findings, it was concluded that horses don’t seem to follow the conventional model of asymmetrical DNA demethylation, because the study didn’t demonstrate evidence of worldwide DNA demethylation of the paternal pronucleus during the first cell cycle.
  • Instead, both parental genomes show sustained and similar levels of methylation and hydroxymethylation throughout pronuclear development. These observations can significantly advance our understanding of the mechanics of genome-wide DNA demethylation in various species.

Cite This Article

APA
Heras S, Smits K, De Schauwer C, Van Soom A. (2017). Dynamics of 5-methylcytosine and 5-hydroxymethylcytosine during pronuclear development in equine zygotes produced by ICSI. Epigenetics Chromatin, 10, 13. https://doi.org/10.1186/s13072-017-0120-x

Publication

Epigenetics & chromatin
ISSN: 1756-8935
NlmUniqueID: 101471619
Country: England
Language: English
Volume: 10
Pages: 13
PII: 13

Researcher Affiliations

Heras, Sonia
  • Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, 9820 Merelbeke, Belgium.
Smits, Katrien
  • Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, 9820 Merelbeke, Belgium.
De Schauwer, Catharina
  • Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, 9820 Merelbeke, Belgium.
Van Soom, Ann
  • Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, 9820 Merelbeke, Belgium.

MeSH Terms

  • 5-Methylcytosine / analogs & derivatives
  • 5-Methylcytosine / metabolism
  • Animals
  • Cell Nucleus / metabolism
  • DNA / metabolism
  • DNA Methylation
  • Histones / metabolism
  • Horses
  • Microscopy, Fluorescence
  • Oocytes / cytology
  • Sperm Injections, Intracytoplasmic
  • Zygote / cytology
  • Zygote / metabolism

References

This article includes 39 references
  1. Reik W, Dean W, Walter J. Epigenetic reprogramming in mammalian development.. Science 2001 Aug 10;293(5532):1089-93.
    pubmed: 11498579doi: 10.1126/science.1063443google scholar: lookup
  2. Dean W, Santos F, Stojkovic M, Zakhartchenko V, Walter J, Wolf E, Reik W. Conservation of methylation reprogramming in mammalian development: aberrant reprogramming in cloned embryos.. Proc Natl Acad Sci U S A 2001 Nov 20;98(24):13734-8.
    doi: 10.1073/pnas.241522698pmc: PMC61110pubmed: 11717434google scholar: lookup
  3. Oswald J, Engemann S, Lane N, Mayer W, Olek A, Fundele R, Dean W, Reik W, Walter J. Active demethylation of the paternal genome in the mouse zygote.. Curr Biol 2000 Apr 20;10(8):475-8.
    doi: 10.1016/S0960-9822(00)00448-6pubmed: 10801417google scholar: lookup
  4. Mayer W, Niveleau A, Walter J, Fundele R, Haaf T. Demethylation of the zygotic paternal genome.. Nature 2000 Feb 3;403(6769):501-2.
    doi: 10.1038/35000656pubmed: 10676950google scholar: lookup
  5. Iqbal K, Jin SG, Pfeifer GP, Szabó PE. Reprogramming of the paternal genome upon fertilization involves genome-wide oxidation of 5-methylcytosine.. Proc Natl Acad Sci U S A 2011 Mar 1;108(9):3642-7.
    doi: 10.1073/pnas.1014033108pmc: PMC3048122pubmed: 21321204google scholar: lookup
  6. Zaitseva I, Zaitsev S, Alenina N, Bader M, Krivokharchenko A. Dynamics of DNA-demethylation in early mouse and rat embryos developed in vivo and in vitro.. Mol Reprod Dev 2007 Oct;74(10):1255-61.
    doi: 10.1002/mrd.20704pubmed: 17290422google scholar: lookup
  7. Xu Y, Zhang JJ, Grifo JA, Krey LC. DNA methylation patterns in human tripronucleate zygotes.. Mol Hum Reprod 2005 Mar;11(3):167-71.
    doi: 10.1093/molehr/gah145pubmed: 15695773google scholar: lookup
  8. Shi W, Dirim F, Wolf E, Zakhartchenko V, Haaf T. Methylation reprogramming and chromosomal aneuploidy in in vivo fertilized and cloned rabbit preimplantation embryos.. Biol Reprod 2004 Jul;71(1):340-7.
    doi: 10.1095/biolreprod.103.024554pubmed: 15028628google scholar: lookup
  9. Jeong YS, Yeo S, Park JS, Koo DB, Chang WK, Lee KK, Kang YK. DNA methylation state is preserved in the sperm-derived pronucleus of the pig zygote.. Int J Dev Biol 2007;51(8):707-14.
    doi: 10.1387/ijdb.072450yjpubmed: 17939117google scholar: lookup
  10. Hou J, Lei TH, Liu L, Cui XH, An XR, Chen YF. DNA methylation patterns in in vitro-fertilised goat zygotes.. Reprod Fertil Dev 2005;17(8):809-13.
    doi: 10.1071/RD05075pubmed: 16476208google scholar: lookup
  11. Beaujean N, Hartshorne G, Cavilla J, Taylor J, Gardner J, Wilmut I, Meehan R, Young L. Non-conservation of mammalian preimplantation methylation dynamics.. Curr Biol 2004 Apr 6;14(7):R266-7.
    doi: 10.1016/j.cub.2004.03.019pubmed: 15062117google scholar: lookup
  12. Tahiliani M, Koh KP, Shen Y, Pastor WA, Bandukwala H, Brudno Y, Agarwal S, Iyer LM, Liu DR, Aravind L, Rao A. Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1.. Science 2009 May 15;324(5929):930-5.
    doi: 10.1126/science.1170116pmc: PMC2715015pubmed: 19372391google scholar: lookup
  13. Kriaucionis S, Heintz N. The nuclear DNA base 5-hydroxymethylcytosine is present in Purkinje neurons and the brain.. Science 2009 May 15;324(5929):929-30.
    doi: 10.1126/science.1169786pmc: PMC3263819pubmed: 19372393google scholar: lookup
  14. Szabó PE, Pfeifer GP. H3K9me2 attracts PGC7 in the zygote to prevent Tet3-mediated oxidation of 5-methylcytosine.. J Mol Cell Biol 2012 Dec;4(6):427-9.
    doi: 10.1093/jmcb/mjs038pubmed: 22750790google scholar: lookup
  15. Wossidlo M, Nakamura T, Lepikhov K, Marques CJ, Zakhartchenko V, Boiani M, Arand J, Nakano T, Reik W, Walter J. 5-Hydroxymethylcytosine in the mammalian zygote is linked with epigenetic reprogramming.. Nat Commun 2011;2:241.
    doi: 10.1038/ncomms1240pubmed: 21407207google scholar: lookup
  16. Salvaing J, Aguirre-Lavin T, Boulesteix C, Lehmann G, Debey P, Beaujean N. 5-Methylcytosine and 5-hydroxymethylcytosine spatiotemporal profiles in the mouse zygote.. PLoS One 2012;7(5):e38156.
    doi: 10.1371/journal.pone.0038156pmc: PMC3364968pubmed: 22693592google scholar: lookup
  17. Li Y, O'Neill C. 5'-Methylcytosine and 5'-hydroxymethylcytosine each provide epigenetic information to the mouse zygote.. PLoS One 2013;8(5):e63689.
    doi: 10.1371/journal.pone.0063689pmc: PMC3653832pubmed: 23691085google scholar: lookup
  18. Salvaing J, Li Y, Beaujean N, O'Neill C. Determinants of valid measurements of global changes in 5'-methylcytosine and 5'-hydroxymethylcytosine by immunolocalisation in the early embryo.. Reprod Fertil Dev 2015 Jun;27(5):755-64.
    pubmed: 25297627doi: 10.1071/rd14136google scholar: lookup
  19. Petrussa L, Van de Velde H, De Rycke M. Similar kinetics for 5-methylcytosine and 5-hydroxymethylcytosine during human preimplantation development in vitro.. Mol Reprod Dev 2016 Jul;83(7):594-605.
    doi: 10.1002/mrd.22656pubmed: 27163211google scholar: lookup
  20. Heras S, Smits K, Leemans B, Van Soom A. Asymmetric histone 3 methylation pattern between paternal and maternal pronuclei in equine zygotes.. Anal Biochem 2015 Feb 15;471:67-9.
    doi: 10.1016/j.ab.2014.11.005pubmed: 25461478google scholar: lookup
  21. Heras S, Forier K, Rombouts K, Braeckmans K, Van Soom A. DNA counterstaining for methylation and hydroxymethylation immunostaining in bovine zygotes.. Anal Biochem 2014 Jun 1;454:14-6.
    doi: 10.1016/j.ab.2014.03.002pubmed: 24631518google scholar: lookup
  22. Li Y, O'Neill C. Persistence of cytosine methylation of DNA following fertilisation in the mouse.. PLoS One 2012;7(1):e30687.
    doi: 10.1371/journal.pone.0030687pmc: PMC3266909pubmed: 22292019google scholar: lookup
  23. Santos F, Hendrich B, Reik W, Dean W. Dynamic reprogramming of DNA methylation in the early mouse embryo.. Dev Biol 2002 Jan 1;241(1):172-82.
    doi: 10.1006/dbio.2001.0501pubmed: 11784103google scholar: lookup
  24. Reis Silva AR, Adenot P, Daniel N, Archilla C, Peynot N, Lucci CM, Beaujean N, Duranthon V. Dynamics of DNA methylation levels in maternal and paternal rabbit genomes after fertilization.. Epigenetics 2011 Aug;6(8):987-93.
    doi: 10.4161/epi.6.8.16073pubmed: 21725201google scholar: lookup
  25. Hyttel P, Greve T, Callesen H. Ultrastructural aspects of oocyte maturation and fertilization in cattle.. J Reprod Fertil Suppl 1989;38:35-47.
    pubmed: 2677348
  26. Barton SC, Arney KL, Shi W, Niveleau A, Fundele R, Surani MA, Haaf T. Genome-wide methylation patterns in normal and uniparental early mouse embryos.. Hum Mol Genet 2001 Dec 15;10(26):2983-7.
    doi: 10.1093/hmg/10.26.2983pubmed: 11751680google scholar: lookup
  27. Heras S, Smits K, Van Soom A. Optimization of DNA methylation immunostaining in equine zygotes produced after ICSI. EpiConcept COST Workshop 2014:1.
  28. Yoshizawa Y, Kato M, Hirabayashi M, Hochi S. Impaired active demethylation of the paternal genome in pronuclear-stage rat zygotes produced by in vitro fertilization or intracytoplasmic sperm injection.. Mol Reprod Dev 2010 Jan;77(1):69-75.
    doi: 10.1002/mrd.21109pubmed: 19743475google scholar: lookup
  29. Zhang P, Su L, Wang Z, Zhang S, Guan J, Chen Y, Yin Y, Gao F, Tang B, Li Z. The involvement of 5-hydroxymethylcytosine in active DNA demethylation in mice.. Biol Reprod 2012 Apr;86(4):104.
    pubmed: 22262693doi: 10.1095/biolreprod.111.096073google scholar: lookup
  30. Li Y, Seah MK, O'Neill C. Mapping global changes in nuclear cytosine base modifications in the early mouse embryo.. Reproduction 2016 Feb;151(2):83-95.
    doi: 10.1530/REP-15-0207pmc: PMC4676261pubmed: 26660107google scholar: lookup
  31. Hahn MA, Qiu R, Wu X, Li AX, Zhang H, Wang J, Jui J, Jin SG, Jiang Y, Pfeifer GP, Lu Q. Dynamics of 5-hydroxymethylcytosine and chromatin marks in Mammalian neurogenesis.. Cell Rep 2013 Feb 21;3(2):291-300.
    doi: 10.1016/j.celrep.2013.01.011pmc: PMC3582786pubmed: 23403289google scholar: lookup
  32. Iurlaro M, Ficz G, Oxley D, Raiber EA, Bachman M, Booth MJ, Andrews S, Balasubramanian S, Reik W. A screen for hydroxymethylcytosine and formylcytosine binding proteins suggests functions in transcription and chromatin regulation.. Genome Biol 2013;14(10):R119.
    doi: 10.1186/gb-2013-14-10-r119pmc: PMC4014808pubmed: 24156278google scholar: lookup
  33. Park JS, Jeong YS, Shin ST, Lee KK, Kang YK. Dynamic DNA methylation reprogramming: active demethylation and immediate remethylation in the male pronucleus of bovine zygotes.. Dev Dyn 2007 Sep;236(9):2523-33.
    doi: 10.1002/dvdy.21278pubmed: 17676637google scholar: lookup
  34. Abdalla H, Hirabayashi M, Hochi S. Demethylation dynamics of the paternal genome in pronuclear-stage bovine zygotes produced by in vitro fertilization and ooplasmic injection of freeze-thawed or freeze-dried spermatozoa.. J Reprod Dev 2009 Aug;55(4):433-9.
    doi: 10.1262/jrd.20229pubmed: 19403998google scholar: lookup
  35. Galli C, Colleoni S, Duchi R, Lagutina I, Lazzari G. Developmental competence of equine oocytes and embryos obtained by in vitro procedures ranging from in vitro maturation and ICSI to embryo culture, cryopreservation and somatic cell nuclear transfer.. Anim Reprod Sci 2007 Mar;98(1-2):39-55.
    doi: 10.1016/j.anireprosci.2006.10.011pubmed: 17101246google scholar: lookup
  36. Polanski Z, Motosugi N, Tsurumi C, Hiiragi T, Hoffmann S. Hypomethylation of paternal DNA in the late mouse zygote is not essential for development.. Int J Dev Biol 2008;52(2-3):295-8.
    doi: 10.1387/ijdb.072347zppubmed: 18311720google scholar: lookup
  37. Smits K, Govaere J, Hoogewijs M, Piepers S, Van Soom A. A pilot comparison of laser-assisted vs piezo drill ICSI for the in vitro production of horse embryos.. Reprod Domest Anim 2012 Feb;47(1):e1-3.
    doi: 10.1111/j.1439-0531.2011.01814.xpubmed: 21950451google scholar: lookup
  38. Galli C, Colleoni S, Duchi R, Lagutina I, Lazzari G. Developmental competence of equine oocytes and embryos obtained by in vitro procedures ranging from in vitro maturation and ICSI to embryo culture, cryopreservation and somatic cell nuclear transfer.. Anim Reprod Sci 2007 Mar;98(1-2):39-55.
    doi: 10.1016/j.anireprosci.2006.10.011pubmed: 17101246google scholar: lookup
  39. Suzuki T, Fujikura K, Higashiyama T, Takata K. DNA staining for fluorescence and laser confocal microscopy.. J Histochem Cytochem 1997 Jan;45(1):49-53.
    doi: 10.1177/002215549704500107pubmed: 9010468google scholar: lookup

Citations

This article has been cited 6 times.
  1. Milazzotto MP, Noonan MJ, de Almeida Monteiro Melo Ferraz M. Mining RNAseq data reveals dynamic metaboloepigenetic profiles in human, mouse and bovine pre-implantation embryos. iScience 2022 Mar 18;25(3):103904.
    doi: 10.1016/j.isci.2022.103904pubmed: 35252810google scholar: lookup
  2. Gastal GDA, Scarlet D, Melchert M, Ertl R, Aurich C. Epigenetic Changes in Equine Embryos after Short-Term Storage at Different Temperatures. Animals (Basel) 2021 May 6;11(5).
    doi: 10.3390/ani11051325pubmed: 34066466google scholar: lookup
  3. Dell'Aquila ME, Asif S, Temerario L, Mastrorocco A, Marzano G, Martino NA, Lacalandra GM, Roelen BA, Carluccio A, Robbe D, Minervini F. Ochratoxin A affects oocyte maturation and subsequent embryo developmental dynamics in the juvenile sheep model. Mycotoxin Res 2021 Feb;37(1):23-37.
    doi: 10.1007/s12550-020-00410-ypubmed: 32996062google scholar: lookup
  4. Zhu Q, Stöger R, Alberio R. A Lexicon of DNA Modifications: Their Roles in Embryo Development and the Germline. Front Cell Dev Biol 2018;6:24.
    doi: 10.3389/fcell.2018.00024pubmed: 29637072google scholar: lookup
  5. Cavalieri V, Spinelli G. Environmental epigenetics in zebrafish. Epigenetics Chromatin 2017 Oct 5;10(1):46.
    doi: 10.1186/s13072-017-0154-0pubmed: 28982377google scholar: lookup
  6. Pang G, Li Y, Shi Q, Tian J, Lou H, Feng Y. Omics sciences for cervical cancer precision medicine from the perspective of the tumor immune microenvironment. Oncol Res 2025;33(4):821-836.
    doi: 10.32604/or.2024.053772pubmed: 40191729google scholar: lookup
Subscribe to our newsletter
Join 99,001 horse owners like you who receive our email updates on horse health and nutrition.