BACKGROUND: Reproductive biology methods rely on in vitro follicle cultures from mature follicles obtained by hormonal stimulation for generating metaphase II oocytes to be fertilised and developed into a healthy embryo. Such techniques are used routinely in both rodent and human species. DNA methylation is a dynamic process that plays a role in epigenetic regulation of gametogenesis and development. In mammalian oocytes, DNA methylation establishment regulates gene expression in the embryos. This regulation is particularly important for a class of genes, imprinted genes, whose expression patterns are crucial for the next generation. The aim of this work was to establish an in vitro culture system for immature mouse oocytes that will allow manipulation of specific factors for a deeper analysis of regulatory mechanisms for establishing transcription regulation-associated methylation patterns. RESULTS: An in vitro culture system was developed from immature mouse oocytes that were grown to germinal vesicles (GV) under two different conditions: normoxia (20% oxygen, 20% O2) and hypoxia (5% oxygen, 5% O2). The cultured oocytes were sorted based on their sizes. Reduced representative bisulphite sequencing (RRBS) and RNA-seq libraries were generated from cultured and compared to in vivo-grown oocytes. In the in vitro cultured oocytes, global and CpG-island (CGI) methylation increased gradually along with oocyte growth, and methylation of the imprinted genes was similar to in vivo-grown oocytes. Transcriptomes of the oocytes grown in normoxia revealed chromatin reorganisation and enriched expression of female reproductive genes, whereas in the 5% O2 condition, transcripts were biased towards cellular stress responses. To further confirm the results, we developed a functional assay based on our model for characterising oocyte methylation using drugs that reduce methylation and transcription. When histone methylation and transcription processes were reduced, DNA methylation at CGIs from gene bodies of grown oocytes presented a lower methylation profile. CONCLUSIONS: Our observations reveal changes in DNA methylation and transcripts between oocytes cultured in vitro with different oxygen concentrations and in vivo-grown murine oocytes. Oocytes grown under 20% O2 had a higher correlation with in vivo oocytes for DNA methylation and transcription demonstrating that higher oxygen concentration is beneficial for the oocyte maturation in ex vivo culture condition. Our results shed light on epigenetic mechanisms for the development of oocytes from an immature to GV oocyte in an in vitro culture model.

Centre for Trophoblast Research University of Cambridge Cambridge CB2 3EG UK

Department of Computer Science King Abdullah 2 School of Information Technology The University of Jordan Amman Jordan

Department of Genetics and Animal Breeding Poznan University of Life Sciences Poznan Poland

Diseases Network Research Unit Faculty of Biochemistry and Molecular Medicine Oulu University Oulu Finland

Epigenetics Program Babraham Institute Cambridge CB22 3AT UK

Laboratory of Early Mammalian Development Department of Molecular Biology and Genetics University of South Bohemia 37005 České Budějovice Czech Republic

School of Medicine Yokohama City University Yokohama Japan

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Obata Y, Kono T. Maternal primary imprinting is established at a specific time for each gene throughout oocyte growth. J Biol Chem. 2002;277(7):5285–5289. doi: 10.1074/jbc.M108586200. PubMed DOI

Hiura H, Obata Y, Komiyama J, Shirai M, Kono T. Oocyte growth-dependent progression of maternal imprinting in mice. Genes Cells. 2006;11(4):353–361. doi: 10.1111/j.1365-2443.2006.00943.x. PubMed DOI

Smallwood SA, Tomizawa S, Krueger F, Ruf N, Carli N, Segonds-Pichon A, Sato S, Hata K, Andrews SR, Kelsey G. Dynamic CpG island methylation landscape in oocytes and preimplantation embryos. Nat Genet. 2011;43(8):811–814. doi: 10.1038/ng.864. PubMed DOI PMC

Veselovska L, Smallwood SA, Saadeh H, Stewart KR, Krueger F, Maupetit-Méhouas S, Arnaud P, Tomizawa S, Andrews S, Kelsey G. Deep sequencing and de novo assembly of the mouse oocyte transcriptome define the contribution of transcription to the DNA methylation landscape. Genome Biol. 2015;16:209. doi: 10.1186/s13059-015-0769-z. PubMed DOI PMC

Ferguson-Smith AC. Genomic imprinting: the emergence of an epigenetic paradigm. Nat Rev Genet. 2011;12(8):565–575. doi: 10.1038/nrg3032. PubMed DOI

Smith ZD, Meissner A. DNA methylation: roles in mammalian development. Nat Rev Genet. 2013;14(3):204–220. doi: 10.1038/nrg3354. PubMed DOI

Gardner DK, Lane M, Stevens J, Schoolcraft WB. Noninvasive assessment of human embryo nutrient consumption as a measure of developmental potential. Fertil Steril. 2001;76(6):1175–1180. doi: 10.1016/S0015-0282(01)02888-6. PubMed DOI

Van Blerkom J, Antczak M, Schrader R. The developmental potential of the human oocyte is related to the dissolved oxygen content of follicular fluid: association with vascular endothelial growth factor levels and perifollicular blood flow characteristics. Hum Reprod. 1997;12(5):1047–1055. doi: 10.1093/humrep/12.5.1047. PubMed DOI

Eppig JJ, Wigglesworth K. Factors affecting the developmental competence of mouse oocytes grown in vitro: oxygen concentration. Mol Reprod Dev. 1995;42(4):447–456. doi: 10.1002/mrd.1080420412. PubMed DOI

Gicquel C, Gaston V, Mandelbaum J, Siffroi J, Flahault A, Le Bouc Y. In vitro fertilization may increase the risk of Beckwith–Wiedemann syndrome related to the abnormal imprinting of the KCN1OT gene. Am J Hum Genet. 2003;72(5):1338–1341. doi: 10.1086/374824. PubMed DOI PMC

Maher ER, Brueton LA, Bowdin SC, Luharia A, Cooper W, Cole TR, Macdonald F, Sampson JR, Barratt CL, Reik W, Hawkins MM. Beckwith–Wiedemann syndrome and assisted reproduction technology (ART) J Med Genet. 2003;40(1):62–64. doi: 10.1136/jmg.40.1.62. PubMed DOI PMC

Gomes MV, Huber J, Ferriani RA, Amaral Neto AM, Ramos ES. Abnormal methylation at the KvDMR1 imprinting control region in clinically normal children conceived by assisted reproductive technologies. Mol Hum Reprod. 2009;15(8):471–477. doi: 10.1093/molehr/gap038. PubMed DOI

Ørstavik KH, Eiklid K, van der Hagen CB, Spetalen S, Kierulf K, Skjeldal O, Buiting K. Another case of imprinting defect in a girl with Angelman syndrome who was conceived by intracytoplasmic semen injection. Am J Hum Genet. 2003;72(1):218–219. doi: 10.1086/346030. PubMed DOI PMC

Cox GF, Bürger J, Lip V, Mau UA, Sperling K, Wu B, Horsthemke B. Intracytoplasmic sperm injection may increase the risk of imprinting defects. Am J Hum Genet. 2002;71(1):162–164. doi: 10.1086/341096. PubMed DOI PMC

DeBaun MR, Niemitz EL, Feinberg AP. Association of in vitro fertilization with Beckwith–Wiedemann syndrome and epigenetic alterations of LIT1 and H19. Am J Hum Genet. 2003;72(1):156–160. doi: 10.1086/346031. PubMed DOI PMC

Ludwig M, Katalinic A, Gross S, Sutcliffe A, Varon R, Horsthemke B. Increased prevalence of imprinting defects in patients with Angelman syndrome born to subfertile couples. J Med Genet. 2005;42(4):289–291. doi: 10.1136/jmg.2004.026930. PubMed DOI PMC

Camprubí C, Iglesias-Platas I, Martin-Trujillo A, Salvador-Alarcon C, Rodriguez MA, Barredo DR, Court F, Monk D. Stability of genomic imprinting and gestational-age dynamic methylation in complicated pregnancies conceived following assisted reproductive technologies. Biol Reprod. 2013;89(3):50. doi: 10.1095/biolreprod.113.108456. PubMed DOI

Sato A, Otsu E, Negishi H, Utsunomiya T, Arima T. Aberrant DNA methylation of imprinted loci in superovulated oocytes. Hum Reprod. 2007;22(1):26–35. doi: 10.1093/humrep/del316. PubMed DOI

Anckaert E, Sánchez F, Billooye K, Smitz J. Dynamics of imprinted DNA methylation and gene transcription for imprinting establishment in mouse oocytes in relation to culture duration variability. Biol Reprod. 2013;89(6):130. doi: 10.1095/biolreprod.113.111641. PubMed DOI

Kuhtz J, Romero S, De Vos M, Smitz J, Haaf T, Anckaert E. Human in vitro oocyte maturation is not associated with increased imprinting error rates at LIT1, SNRPN, PEG3 and GTL2. Hum Reprod. 2014;29(9):1995–2005. doi: 10.1093/humrep/deu155. PubMed DOI

Kobayashi H, Sakurai T, Imai M, Takahashi N, Fukuda A, Yayoi O, Sato S, Nakabayashi K, Hata K, Sotomaru Y, Suzuki Y, Kono T. Contribution of intragenic DNA methylation in mouse gametic DNA methylomes to establish oocyte-specific heritable marks. PLoS Genet. 2012;8(1):e1002440. doi: 10.1371/journal.pgen.1002440. PubMed DOI PMC

Shirane K, Toh H, Kobayashi H, Miura F, Chiba H, Ito T, Kono T, Sasaki H. Mouse oocyte methylomes at base resolution reveal genome-wide accumulation of non-CpG methylation and role of DNA methyltransferases. PLoS Genet. 2013;9(4):e1003439. doi: 10.1371/journal.pgen.1003439. PubMed DOI PMC

Gahurova L, Tomizawa S, Smallwood SA, Stewart-Morgan KR, Saadeh H, Kim J, Andrews SR, Chen T, Kelsey G. Transcription and chromatin determinants of de novo DNA methylation timing in oocytes. Epigenetics Chromatin. 2017;10:25. doi: 10.1186/s13072-017-0133-5. PubMed DOI PMC

Honda A, Hirose M, Inoue K, Hiura H, Miki H, Ogonuki N, Sugimoto M, Abe K, Kanatsu-Shinohara M, Kono T, Shinohara T, Ogura A. Large-scale production of growing oocytes in vitro from neonatal mouse ovaries. Int J Dev Biol. 2009;53(4):605–613. doi: 10.1387/ijdb.082607ah. PubMed DOI

Warzych E, Peippo J, Szydlowski M, Lechniak D. Supplements to in vitro maturation media affect the production of bovine blastocysts and their apoptotic index but not the proportions of matured and apoptotic oocytes. Anim Reprod Sci. 2007;97(3–4):334–343. doi: 10.1016/j.anireprosci.2006.01.011. PubMed DOI

Jamnongjit M, Gill A, Hammes SR. Epidermal growth factor receptor signaling is required for normal ovarian steroidogenesis and oocyte maturation. Proc Natl Acad Sci USA. 2005;102(45):16257–16262. doi: 10.1073/pnas.0508521102. PubMed DOI PMC

Eppig JJ, O’Brien MJ, Pendola FL, Watanabe S. Factors affecting the developmental competence of mouse oocytes grown in vitro: follicle-stimulating hormone and insulin. Biol Reprod. 1998;59(6):1445–1453. doi: 10.1095/biolreprod59.6.1445. PubMed DOI

Catt JW, Henman M. Toxic effects of oxygen on human embryo development. Hum Reprod. 2000;15(Suppl 2):199–206. doi: 10.1093/humrep/15.suppl_2.199. PubMed DOI

Pierce KE, Grunvald EL, Schultz RM, Kopf GS. Temporal pattern of synthesis of the mouse cortical granule protein, p75, during oocyte growth and maturation. Dev Biol. 1992;152(1):145–151. doi: 10.1016/0012-1606(92)90164-C. PubMed DOI

Zuccotti M, Giorgi Rossi P, Martinez A, Garagna S, Forabosco A, Redi CA. Meiotic and developmental competence of mouse antral oocytes. Biol Reprod. 1998;58(3):700–704. doi: 10.1095/biolreprod58.3.700. PubMed DOI

Meissner A, Gnirke A, Bell GW, Ramsahoye B, Lander ES, Jaenisch R. Reduced representation bisulfite sequencing for comparative high-resolution DNA methylation analysis. Nucleic Acids Res. 2005;33(18):5868–5877. doi: 10.1093/nar/gki901. PubMed DOI PMC

Illingworth RS, Gruenewald-Schneider U, Webb S, Kerr ARW, James KD, Turner DJ, Smith C, Harrison DJ, Andrews R, Bird AP. Orphan CpG islands identify numerous conserved promoters in the mammalian genome. PLoS Genet. 2010;6(9):e1001134. doi: 10.1371/journal.pgen.1001134. PubMed DOI PMC

Jagarlamudi K, Rajkovic A. Oogenesis: transcriptional regulators and mouse models. Mol Cell Endocrinol. 2012;356(1–2):31–39. doi: 10.1016/j.mce.2011.07.049. PubMed DOI

Li KK, Luo C, Wang D, Jiang H, Zheng YG. Chemical and biochemical approaches in the study of histone methylation and demethylation. Med Res Rev. 2012;32(4):815–867. doi: 10.1002/mrr.20228. PubMed DOI PMC

Li L, Ji S, Yang J, Li X, Zhang J, Zhang Y, Hu Z, Liu Y. Wnt/β-catenin signaling regulates follicular development by modulating the expression of Foxo3a signaling components. Mol Cell Endocrinol. 2014;382(2):915–925. doi: 10.1016/j.mce.2013.11.007. PubMed DOI

Shi Y, Lan F, Matson C, Mulligan P, Whetstine JR, Cole PA, Casero RA, Shi Y. Histone demethylation mediated by the nuclear amine oxidase homolog LSD1. Cell. 2004;119(7):941–953. doi: 10.1016/j.cell.2004.12.012. PubMed DOI

Thinnes CC, England KS, Kawamura A, Chowdhury R, Schofield CJ, Hopkinson RJ. Targeting histone lysine demethylases—progress, challenges, and the future. Biochim Biophys Acta. 2014;1839(12):1416–1432. doi: 10.1016/j.bbagrm.2014.05.009. PubMed DOI PMC

Stewart KR, Veselovska L, Kim J, Huang J, Saadeh H, Tomizawa S, Smallwood SA, Chen T, Kelsey G. Dynamic changes in histone modifications precede de novo DNA methylation in oocytes. Genes Dev. 2015;29(23):2449–2462. doi: 10.1101/gad.271353.115. PubMed DOI PMC

Ciccone DN, Su H, Hevi S, Gay F, Lei H, Bajko J, Xu G, Li E, Chen T. KDM1B is a histone H3K4 demethylase required to establish maternal genomic imprints. Nature. 2009;461(7262):415–418. doi: 10.1038/nature08315. PubMed DOI

Noh K, Wang H, Kim HR, Wenderski W, Fang F, Li CH, Dewell S, Hughes SH, Melnick AM, Patel DJ, Li H, Allis CD. Engineering of a histone-recognition domain in dnmt3a alters the epigenetic landscape and phenotypic features of mouse ESCs. Mol Cell. 2015;59(1):89–103. doi: 10.1016/j.molcel.2015.05.017. PubMed DOI PMC

Peat JR, Dean W, Clark SJ, Krueger F, Smallwood SA, Ficz G, Kim JK, Marioni JC, Hore TA, Reik W. Genome-wide bisulfite sequencing in zygotes identifies demethylation targets and maps the contribution of TET3 oxidation. Cell Rep. 2014;9(6):1990–2000. doi: 10.1016/j.celrep.2014.11.034. PubMed DOI PMC

Hayashi K, Ogushi S, Kurimoto K, Shimamoto S, Ohta H, Saitou M. Offspring from oocytes derived from in vitro primordial germ cell-like cells in mice. Science. 2012;338(6109):971–975. doi: 10.1126/science.1226889. PubMed DOI

Morohaku K, Tanimoto R, Sasaki K, Kawahara-Miki R, Kono T, Hayashi K, Hirao Y, Obata Y. Complete in vitro generation of fertile oocytes from mouse primordial germ cells. Proc Natl Acad Sci USA. 2016;113(32):9021–9026. doi: 10.1073/pnas.1603817113. PubMed DOI PMC

Saenz-de-Juano MD, Ivanova E, Billooye K, Herta A, Smitz J, Kelsey G, Anckaert E. Genome-wide assessment of DNA methylation in mouse oocytes reveals effects associated with in vitro growth, superovulation, and sexual maturity. Clin Epigenet. 2019;11(1):197. doi: 10.1186/s13148-019-0794-y. PubMed DOI PMC

Sánchez F, Adriaenssens T, Romero S, Smitz J. Quantification of oocyte-specific transcripts in follicle-enclosed oocytes during antral development and maturation in vitro. Mol Hum Reprod. 2009;15(9):539–550. doi: 10.1093/molehr/gap051. PubMed DOI

Chotalia M, Smallwood SA, Ruf N, Dawson C, Lucifero D, Frontera M, James K, Dean W, Kelsey G. Transcription is required for establishment of germline methylation marks at imprinted genes. Genes Dev. 2009;23(1):105–117. doi: 10.1101/gad.495809. PubMed DOI PMC

Brind’Amour J, Kobayashi H, Albert JR, Shirane K, Sakashita A, Kamio A, Bogutz A, Koike T, Karimi MM, Lefebvre L, Kono T, Lorincz MC. LTR retrotransposons transcribed in oocytes drive species-specific and heritable changes in DNA methylation. Nat Commun. 2018;9(1):1–14. doi: 10.1038/s41467-018-05841-x. PubMed DOI PMC

Sendžikaitė G, Kelsey G. The role and mechanisms of DNA methylation in the oocyte. Essays in biochem. 2019;63(6):691–705. doi: 10.1042/EBC20190043. PubMed DOI PMC

Nakagawa K, Shirai A, Nishi Y, Sugiyama R, Kuribayashi Y, Sugiyama R, Inoue M. A study of the effect of an extremely low oxygen concentration on the development of human embryos in assisted reproductive technology. Reprod Med Biol. 2010;9(3):163–168. doi: 10.1007/s12522-010-0052-7. PubMed DOI PMC

de los Santos MJ, Gámiz P, Albert C, Galán A, Viloria T, Pérez S, Romero JL, Remohï J. Reduced oxygen tension improves embryo quality but not clinical pregnancy rates: a randomized clinical study into ovum donation cycles. Fertil Steril. 2013;100(2):402–407. doi: 10.1016/j.fertnstert.2013.03.044. PubMed DOI

Van Montfoort APA, Arts EGJM, Wijnandts L, Sluijmer A, Pelinck M, Land JA, Van Echten-Arends J. Reduced oxygen concentration during human IVF culture improves embryo utilization and cumulative pregnancy rates per cycle. Hum Reprod Open. 2020;2020(1):36. PubMed PMC

Fernandez-Twinn DS, Hjort L, Novakovic B, Ozanne SE, Saffery R. Intrauterine programming of obesity and type 2 diabetes. Diabetologia. 2019;62(10):1789–1801. doi: 10.1007/s00125-019-4951-9. PubMed DOI PMC

Lee MG, Wynder C, Schmidt DM, McCafferty DG, Shiekhattar R. Histone H3 lysine 4 demethylation is a target of nonselective antidepressive medications. Chem Biol. 2006;13(6):563–567. doi: 10.1016/j.chembiol.2006.05.004. PubMed DOI

Krueger F, Andrews SR. Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications. Bioinformatics. 2011;27(11):1571–1572. doi: 10.1093/bioinformatics/btr167. PubMed DOI PMC

Proudhon C, Duffié R, Ajjan S, Cowley M, Iranzo J, Carbajosa G, Saadeh H, Holland ML, Oakey RJ, Rakyan VK, Schulz R, Bourc'his D. Protection against de novo methylation is instrumental in maintaining parent-of-origin methylation inherited from the gametes. Mol Cell. 2012;47(6):909–920. doi: 10.1016/j.molcel.2012.07.010. PubMed DOI PMC

Santos F, Hendrich B, Reik W, Dean W. Dynamic reprogramming of DNA methylation in the early mouse embryo. Dev Biol. 2002;241(1):172–182. doi: 10.1006/dbio.2001.0501. PubMed DOI

Burgess A, Vigneron S, Brioudes E, Labbé J, Lorca T, Castro A. Loss of human Greatwall results in G2 arrest and multiple mitotic defects due to deregulation of the cyclin B-Cdc2/PP2A balance. Proc Natl Acad Sci USA. 2010;107(28):12564–12569. doi: 10.1073/pnas.0914191107. PubMed DOI PMC

An Interplay between Epigenetics and Translation in Oocyte Maturation and Embryo Development: Assisted Reproduction Perspective