University of Oulu

Naillat, F., Saadeh, H., Nowacka-Woszuk, J. et al. Oxygen concentration affects de novo DNA methylation and transcription in in vitro cultured oocytes. Clin Epigenet 13, 132 (2021).

Oxygen concentration affects de novo DNA methylation and transcription in in vitro cultured oocytes

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Author: Naillat, Florence1,2; Saadeh, Heba1,3; Nowacka-Woszuk, Joanna1,4;
Organizations: 1Epigenetics Program, Babraham Institute, Cambridge, CB22 3AT, UK
2Diseases Network Research Unit, Faculty of Biochemistry and Molecular Medicine, Oulu University, Oulu, Finland
3Department of Computer Science, King Abdullah II School of Information Technology, The University of Jordan, Amman, Jordan
4Department of Genetics and Animal Breeding, Poznan University of Life Sciences, Poznan, Poland
5Laboratory of Early Mammalian Development, Department of Molecular Biology and Genetics, University of South Bohemia, 37005, České Budějovice, Czech Republic
6School of Medicine, Yokohama City University, Yokohama, Japan
7Centre for Trophoblast Research, University of Cambridge, Cambridge, CB2 3EG, UK
Format: article
Version: published version
Access: open
Online Access: PDF Full Text (PDF, 2.3 MB)
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Language: English
Published: Springer Nature, 2021
Publish Date: 2021-11-01


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.

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Series: Clinical epigenetics
ISSN: 1868-7075
ISSN-E: 1868-7083
ISSN-L: 1868-7075
Volume: 13
Issue: 1
Article number: 132
DOI: 10.1186/s13148-021-01116-3
Type of Publication: A1 Journal article – refereed
Field of Science: 3111 Biomedicine
Funding: This work was supported by grants for FN from the Academy of Finland Profiling funding to the University of Oulu Profi3 (311934), Academy of Finland post-doctoral Fellowship (243014583), Foundations’ Post Doc Pool (Svenska Kulturfonden), and Finnish Cultural Foundation (Pekka ja Jukka-Pekka Lylykarin rahasto); for JNW Polish Ministry of Science and Higher Education post-doctoral fellowship (Mobility Plus Programme, No: 609/MOB/2011/0); and for G.K. by the UK Biotechnology and Biological Sciences Research Council and Medical Research Council (Grants G0800013 and MR/K011332/1).
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