Dynamics of Imprinted DNA Methylation and Gene Transcription for Imprinting Establishment in Mouse Oocytes in Relation to Culture Duration Variability1

Anckaert, Ellen; Sánchez, Flor; Billooye, Katy; Smitz, Johan

doi:10.1095/biolreprod.113.111641

Cited by 11 publications

(8 citation statements)

References 42 publications

(62 reference statements)

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“…In our analysis, we did not detect methylation alterations at genes known to be involved in methylation establishment and maintenance, consistent with no significant effect of ART or sexual maturity on their expression. These results are consistent with previous findings of similar transcript abundance for a subset of these genes in in vivo and in vitro developed oocytes [26, 34]. Further experiments will need to evaluate whether loss of gDMR methylation in blastocysts could be related to altered protein abundance or nuclear localization of those factors.…”

Section: Discussionsupporting

confidence: 90%

Genome-wide assessment of DNA methylation in mouse oocytes reveals effects associated with in vitro growth, superovulation, and sexual maturity

et al. 2019

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BackgroundIn vitro follicle culture (IFC), as applied in the mouse system, allows the growth and maturation of a large number of immature preantral follicles to become mature and competent oocytes. In the human oncofertility clinic, there is increasing interest in developing this technique as an alternative to ovarian cortical tissue transplantation and to preserve the fertility of prepubertal cancer patients. However, the effect of IFC and hormonal stimulation on DNA methylation in the oocyte is not fully known, and there is legitimate concern over epigenetic abnormalities that could be induced by procedures applied during assisted reproductive technology (ART).ResultsIn this study, we present the first genome-wide analysis of DNA methylation in MII oocytes obtained after natural ovulation, after IFC and after superovulation. We also performed a comparison between prepubertal and adult hormonally stimulated oocytes. Globally, the distinctive methylation landscape of oocytes, comprising alternating hyper- and hypomethylated domains, is preserved irrespective of the procedure. The conservation of methylation extends to the germline differential methylated regions (DMRs) of imprinted genes, necessary for their monoallelic expression in the embryo. However, we do detect specific, consistent, and coherent differences in DNA methylation in IFC oocytes, and between oocytes obtained after superovulation from prepubertal compared with sexually mature females. Several methylation differences span entire transcription units. Among these, we found alterations in Tcf4, Sox5, Zfp521, and other genes related to nervous system development.ConclusionsOur observations show that IFC is associated with altered methylation at specific set of loci. DNA methylation of superovulated prepubertal oocytes differs from that of superovulated adult oocytes, whereas oocytes from superovulated adult females differ very little from naturally ovulated oocytes. Importantly, we show that regions other than imprinted gDMRs are susceptible to methylation changes associated with superovulation, IFC, and/or sexual immaturity in mouse oocytes. Our results provide an important reference for the use of in vitro growth and maturation of oocytes, particularly from prepubertal females, in assisted reproductive treatments or fertility preservation.

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Section: Discussionsupporting

confidence: 90%

Genome-wide assessment of DNA methylation in mouse oocytes reveals effects associated with in vitro growth, superovulation, and sexual maturity

et al. 2019

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“…In our study, the two different oxygen conditions (20% vs. 5% O 2 ) did not affect the temporal acquisition of correct imprinted DNA methylation patterns in the denuded oocytes, as the methylation of the imprinted genes was closer to the class IV in vivo oocytes as the igDMR methylation was restored in in vitro 5% and 20% O 2 oocytes. This result is in line with previous studies using in vitro culture model or super-ovulation method demonstrating that imprinted DNA methylation acquisition in oocytes is a robust mechanism and is not influenced by culture conditions [ 19 , 44 ]. Imprinted genes are regulated by the methylation status of the imprinting control region (ICR), and when the methylation of the ICR failed due to the loss of transcription, it results in aberrant methylation of the igDMRs [ 46 ].…”

Section: Discussionsupporting

confidence: 91%

“…Similar controversial results have been published in relation to super-ovulation. Super-ovulated oocytes from mice and humans show that Peg1 and H19 imprinted genes were differentially methylated in super-ovulated oocytes compared to normally ovulating females, suggesting a link between methylation of imprinted genes and super-ovulation [ 18 ], whereas other studies reveal no effect on the methylation of the imprinted genes by super-ovulation [ 19 , 20 ].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2021

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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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“…A recent study demonstrated no significant changes in the total mRNA amount for the ME genes Dnmt3a , Dnmt3l and Zfp57 in preovulatory-aged IVM oocytes in the mouse [47] . In line with this, we also did not observe a change in Zfp57 expression after in vitro preovulatory aging.…”

Section: Discussionmentioning

confidence: 95%

Pre- and Postovulatory Aging of Murine Oocytes Affect the Transcript Level and Poly(A) Tail Length of Maternal Effect Genes

et al. 2014

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Maternal effect genes code for oocyte proteins that are important for early embryogenesis. Transcription in oocytes does not take place from the onset of meiotic progression until zygotic genome activation. During this period, protein levels are regulated posttranscriptionally, for example by poly(A) tail length. Posttranscriptional regulation may be impaired in preovulatory and postovulatory aged oocytes, caused by delayed ovulation or delayed fertilization, respectively, and may lead to developmental defects. We investigated transcript levels and poly(A) tail length of ten maternal effect genes in in vivo- and in vitro- (follicle culture) grown oocytes after pre- and postovulatory aging. Quantitative RT-PCR was performed using random hexamer-primed cDNA to determine total transcript levels and oligo(dT)16-primed cDNA to analyze poly(A) tail length. Transcript levels of in vivo preovulatory-aged oocytes remained stable except for decreases in Brg1 and Tet3. Most genes investigated showed a tendency towards increased poly(A) content. Polyadenylation of in vitro preovulatory-aged oocytes was also increased, along with transcript level declines of Trim28, Nlrp2, Nlrp14 and Zar1. In contrast to preovulatory aging, postovulatory aging of in vivo- and in vitro-grown oocytes led to a shortening of poly(A) tails. Postovulatory aging of in vivo-grown oocytes resulted in deadenylation of Nlrp5 after 12 h, and deadenylation of 4 further genes (Tet3, Trim28, Dnmt1, Oct4) after 24 h. Similarly, transcripts of in vitro-grown oocytes were deadenylated after 12 h of postovulatory aging (Tet3, Trim28, Zfp57, Dnmt1, Nlrp5, Zar1). This impact of aging on poly(A) tail length may affect the timed translation of maternal effect gene transcripts and thereby contribute to developmental defects.

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Dynamics of Imprinted DNA Methylation and Gene Transcription for Imprinting Establishment in Mouse Oocytes in Relation to Culture Duration Variability1

Cited by 11 publications

References 42 publications

Genome-wide assessment of DNA methylation in mouse oocytes reveals effects associated with in vitro growth, superovulation, and sexual maturity

Genome-wide assessment of DNA methylation in mouse oocytes reveals effects associated with in vitro growth, superovulation, and sexual maturity

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

Pre- and Postovulatory Aging of Murine Oocytes Affect the Transcript Level and Poly(A) Tail Length of Maternal Effect Genes

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