Limiting dilution bisulfite (pyro)sequencing reveals parent-specific methylation patterns in single early mouse embryos and bovine oocytes

Hajj, Nady El; Trapphoff, Tom; Linke, Matthias; May, Andreas; Hansmann, T.; Kuhtz, Juliane; Reifenberg, Kurt; Heinzmann, Julia; Niemann, Heiner; Daser, Angelika; Eichenlaub-Ritter, Ursula; Zechner, Ulrich; Haaf, Thomas

doi:10.4161/epi.6.10.17202

Cited by 53 publications

(48 citation statements)

References 38 publications

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“…None of 258 DNA molecules from immature and of 296 from in vitro matured oocytes showed O50% methylation, indicating that epimutations (at least in the three studied genes) are extremely rare or lack completely in the analyzed groups. Consistent with our recent studies (El Hajj et al 2011, the rate of single methylated CpG sites in the three studied genes varied between 0 and 2.4% (data not shown). No significant differences (PO0.05) existed between the different oocyte groups.…”

Section: Gene-specific Methylationsupporting

confidence: 77%

“…We applied the recently developed and validated limiting dilution approach and direct bisulfite sequencing (El Hajj et al 2011 to analyze the methylation profile of individual DNA molecules in three nonimprinted genes, including solute carrier family 2 member 1 (SLC2A1/facilitated glucose transporter), peroxiredoxin 1 (PRDX1), and zygotic arrest gene 1 (ZAR1) from prepubertal and adult oocytes, collected from donors with and without treatment with FSH.…”

Section: Introductionmentioning

confidence: 99%

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DNA methylation and mRNA expression profiles in bovine oocytes derived from prepubertal and adult donors

Diederich¹,

Hansmann²,

Heinzmann³

et al. 2012

Reproduction

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The developmental capacity of oocytes from prepubertal cattle is reduced compared with their adult counterparts, and epigenetic mechanisms are thought to be involved herein. Here, we analyzed DNA methylation in three developmentally important, nonimprinted genes (SLC2A1, PRDX1, ZAR1) and two satellite sequences, i.e. 'bovine testis satellite I' (BTS) and 'Bos taurus alpha satellite I' (BTaS). In parallel, mRNA expression of the genes was determined by quantitative real-time PCR. Oocytes were retrieved from prepubertal calves and adult cows twice per week over a 3-week period by ultrasound-guided follicular aspiration after treatment with FSH and/or IGF1. Both immature and in vitro matured prepubertal and adult oocytes showed a distinct hypomethylation profile of the three genes without differences between the two types of donors. The methylation status of the BTS sequence changed according to the age and treatment while the methylation status of BTaS sequence remained largely unchanged across the different age and treatment groups. Relative transcript abundance of the selected genes was significantly different in immature and in vitro matured oocytes; only minor changes related to origin and treatment were observed. In conclusion, methylation levels of the investigated satellite sequences were high (O50%) in all groups and showed significant variation depending on the age, treatment, or in vitro maturation. To what extent this is involved in the acquisition of developmental competence of bovine oocytes needs further study.

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Section: Gene-specific Methylationsupporting

confidence: 77%

Section: Introductionmentioning

confidence: 99%

DNA methylation and mRNA expression profiles in bovine oocytes derived from prepubertal and adult donors

Diederich¹,

Hansmann²,

Heinzmann³

et al. 2012

Reproduction

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“…Data from Patrushev &Minkevich (2008) andCarrell (2012). recovered from superovulated (7.5 IU eCG/hCG) females had paternal H19 loss of methylation (LOM, Box 3) in 2/10 embryos, maternal Snrpn LOM in 2/10 embryos, and maternal H19 gain of methylation (GOM) in 1/10 embryos. This frequency of imprinting errors was not statistically different from controls (El Hajj et al 2011). However, as 12-24% of DNA strands per gene per embryo were recovered, additional perturbations may have been missed.…”

Section: Ovarian Stimulationmentioning

confidence: 76%

Genomic imprints as a model for the analysis of epigenetic stability during assisted reproductive technologies

Denomme¹,

Mann²

2012

Reproduction

112

118

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Gamete and early embryo development are important stages when genome-scale epigenetic transitions are orchestrated. The apparent lack of remodeling of differential imprinted DNA methylation during preimplantation development has lead to the argument that epigenetic disruption by assisted reproductive technologies (ARTs) is restricted to imprinted genes. We contend that aberrant imprinted methylation arising from assisted reproduction or infertility may be an indicator of more global epigenetic instability. Here, we review the current literature on the effects of ARTs, including ovarian stimulation, in vitro oocyte maturation, oocyte cryopreservation, IVF, ICSI, embryo culture, and infertility on genomic imprinting as a model for evaluating epigenetic stability. Undoubtedly, the relationship between impaired fertility, ARTs, and epigenetic stability is unquestionably complex. What is clear is that future studies need to be directed at determining the molecular and cellular mechanisms giving rise to epigenetic errors.

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“…Intracytoplasmic morphologically selected sperm injection (IMSI) requires a much higher magnification (»6000£) for better morphological assessment. 28 We employed limiting dilution (LD) bisulfite pyrosequencing 26 to analyze single-allele methylation of one paternally methylated (GTL2) and 2 maternally methylated (LIT1 and PEG3) imprinted genes in 4 different classes of sperm from fertile donors: IMSIC are the "best" sperm selected under a high-powered microscope. They display normal morphology and do not contain any vacuoles.…”

Section: Resultsmentioning

confidence: 99%

“…Since it is usually the density of methylated CpGs rather than individual CpGs that turns a gene "on" or "off," only alleles with the majority of CpGs being aberrantly (de)methylated (allele methylation errors) are considered as epimutations, whereas single CpG errors are most likely without functional consequences. 25,26 Plasmid bisulfite sequencing of a limited number of sperm DNA alleles suggests the existence of true epimutations of both paternally (e.g., H19) and maternally methylated alleles (e.g., MEST) in sperm of infertile men. 15,27 Here we employed different techniques for in depth methylation analysis of individual DNA molecules in different classes of sperm.…”

Section: Introductionmentioning

confidence: 99%

Epigenetic heterogeneity of developmentally important genes in human sperm: Implications for assisted reproduction outcome

Kuhtz¹,

Schneider²,

Hajj³

et al. 2014

Epigenetics

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The molecular basis of male infertility is poorly understood, the majority of cases remaining unsolved. The association of aberrant sperm DNA methylation patterns and compromised semen parameters suggests that disturbances in male germline epigenetic reprogramming contribute to this problem. So far there are only few data on the epigenetic heterogeneity of sperm within a given sample and how to select the best sperm for successful infertility treatment. Limiting dilution bisulfite sequencing of small pools of sperm from fertile donors did not reveal significant differences in the occurrence of abnormal methylation imprints between sperm with and without morphological abnormalities. Intracytoplasmic morphologically selected sperm injection was not associated with an improved epigenetic quality, compared to standard intracytoplasmatic sperm injection. Deep bisulfite sequencing (DBS) of 2 imprinted and 2 pluripotency genes in sperm from men attending a fertility center showed that in both samples with normozoospermia and oligoasthenoteratozoospermia (OAT) the vast majority of sperm alleles was normally (de) methylated and the percentage of epimutations (allele methylation errors) was generally low (<1%). However, DBS allowed one to identify and quantify these rare epimutations with high accuracy. Sperm samples not leading to a pregnancy, in particular in the OAT group, had significantly more epimutations in the paternally methylated GTL2 gene than samples leading to a live birth. All 13 normozoospermic and 13 OAT samples leading to a child had <1% GTL2 epimutations, whereas one (7%) of 14 normozoospermic and 7 (50%) of 14 OAT samples without pregnancy displayed 1-14% GTL2 epimutations.

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Limiting dilution bisulfite (pyro)sequencing reveals parent-specific methylation patterns in single early mouse embryos and bovine oocytes

Cited by 53 publications

References 38 publications

DNA methylation and mRNA expression profiles in bovine oocytes derived from prepubertal and adult donors

DNA methylation and mRNA expression profiles in bovine oocytes derived from prepubertal and adult donors

Genomic imprints as a model for the analysis of epigenetic stability during assisted reproductive technologies

Epigenetic heterogeneity of developmentally important genes in human sperm: Implications for assisted reproduction outcome

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