Reproductive and Developmental Medicine

: 2017  |  Volume : 1  |  Issue : 1  |  Page : 13--17

Epigenetic Modification in Oocyte and Preimplantation Embryonic Development

Yi-Xin Ren1, William Chang1, Jie Qiao2,  
1 Department of Obstetrics and Gynecology, Peking University Third Hospital; Key Laboratory of Assisted Reproduction, Ministry of Education; Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproductive Technology, Beijing 100191, China
2 Department of Obstetrics and Gynecology, Peking University Third Hospital; Key Laboratory of Assisted Reproduction, Ministry of Education; Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproductive Technology, Beijing 100191; Beijing Advanced Innovation Center for Genomics, Beijing 100871; Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China

Correspondence Address:
Jie Qiao
Department of Obstetrics and Gynecology, Peking University Third Hospital, No. 49 North Garden Road, Haidian District, Beijing 100191


DNA methylation and histone modification are two of the most characterized epigenetic modifications. With advanced detecting techniques, particularly single-cell sequencing, we can dissect epigenomic patterns and their regulatory roles in the growth and differentiation of gametes and early embryos in animals and humans. Assisted reproductive technology (ART) procedures have been shown to influence the methylation of certain genes. Aberrant epigenetic regulation may cause several developmental disorders and clinical diseases. Here, we describe some concepts in epigenetics and review recent researches on DNA methylation and the histone modification profile and their regulatory roles during early embryo development. We also summarize the recent progress in understanding the imprinting disorders associated with ART procedures.

How to cite this article:
Ren YX, Chang W, Qiao J. Epigenetic Modification in Oocyte and Preimplantation Embryonic Development.Reprod Dev Med 2017;1:13-17

How to cite this URL:
Ren YX, Chang W, Qiao J. Epigenetic Modification in Oocyte and Preimplantation Embryonic Development. Reprod Dev Med [serial online] 2017 [cited 2021 May 11 ];1:13-17
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Life begins at fertilization, when highly specific sperm and egg cells meet and form a totipotent zygote, which further develops into a new individual.[1] Events that occur during embryonic development, including zygotic genome activation (ZGA), cleavage, and embryonic cell differentiation, rely on highly coordinated and dynamic regulation of genomes, transcriptomes, and epigenomes.[2] With the development of advanced techniques, such as high-throughput sequencing, we can detect the epigenetic modifications that occur in the first few days or even few hours of a new life.[3]

The term “Epigenetic” was first proposed by Waddington 70 years ago to explain the connection between phenotype and genotype during development.[4] Epigenetics is defined as inheritable functional changes that do not involve changes in the DNA sequence of a gene.[5] Although cells in different organs and tissues share the same DNA sequence, they may have differential expression profiles.[6] Epigenetic modification plays an important role in genomic reprogramming and the establishment of early embryonic development patterns.[7],[8] During cell proliferation and differentiation, epigenetic modification regulates gene expression, forming barriers that keep the cell typespecification in a one-way process.[3] Mechanisms involved in epigenetic inheritance include self-sustaining feedback loops; chromatin-based mechanisms such as DNA methylation, histone variants, and histone modifications; noncoding and coding RNA such as maternal stored mRNAs, long noncoding RNAs, and small RNAs; and structural templating (prions and chaperones).[9] Among these mechanisms, DNA methylation and histone modification are the best-studied epigenetic mechanisms for transgenerational inheritance. In this review, we will focus on the recent studies on DNA methylation and histone modification during the early embryonic development.

 DNA Methylation in Embryonic Development

5-methylcytosine (5mC) is the methylated form of the fifth carbon of cytosine, which is considered the “fifth base of DNA” due to its importance in controlling gene regulation.[10] In the early 1980s, researchers used high-performance liquid chromatography to analyze the 5mC content in human somatic cells and found that the amount and distribution of 5mC were in different tissues and cells, with the 5mC content ranging from 0.76% to 1%.[11] CpG islands are about 1,000 bp long regions in the genome with a high density of CpG sites.[12] In mammals, approximately 60%-80% of CpG sites are methylated. However, about 10% of CpGs in CpG islands are frequently hypomethylated, and these hypomethylated CpGs are associated with gene promoters. More than 70% of promoter regions have a high CpG content.[13],[14] The effects of DNA methylation include transcription repression (by inhibiting the transcription factor binding or recruiting methyl-binding proteins and their associated chromatin remodeling factors), gene imprinting, X chromosome inactivation, and the suppression of parasitic DNA sequences.[15],[16]

In mammals, the maintenance of methylation in the genome depends on the activity of DNA methyltransferase (Dnmt).[17] At least five Dnmts have been identified: Dnmt1, Dnmt2, Dnmt3A, Dnmt3B, and Dnmt3L.[18]Dnmt1o, Dnmt1s, and Dnmt1p are three isoforms of Dnmt1 transcripts that are produced by the alternative splicing of the first exon.[19]Dnmt1o is expressed in mouse oocytes and preimplantation embryos. Dnmt1s is expressed in somatic cells, while Dnmt1p is only found in pachytene spermatocytes.[19] Target depletion of Dnmt1o in female mice led to miscarriage during the last third of pregnancy. Furthermore, embryos derived from such oocytes displayed a loss of methylation at some imprinted loci and allele-specific expression.[17] In the Dnmt1-deficient mouse embryos, random X-chromosome inactivation was unstable as a consequence of hypomethylation, indicating that methylation is required for the maintenance of X inactivation in embryos.[16] On the other hand, overexpression of Dnmt1 caused abnormal hypermethylation in the genome, loss of imprinting, and death of the embryo.[20] Using high-resolution sequencing of hairpin-bisulfite amplicons to study DNA methylation, Arand et al. found considerable increases in de novo methylation activity of Dnmt1 at certain repetitive elements and single copy sequences in embryonic stem cells.[21] Moreover, Dnmt3a and Dnmt3b are associated with de novo methylation and maintenance of methylation.[21] In human preimplantation embryos, Dnmt3b contributed to demethylation in blastocysts.[22]

DNA methylation is more dynamic in germ line cells and early embryos than it is in somatic cells.[23] Due to the limited availability of embryos, it was difficult to directly draw a whole-genome methylation map of preimplantation embryos. However, using indirect immunofluorescence with an anti-5-methyl-cytosine antibody, researchers can observe the methylation patterns in embryos. Immediately after fertilization, the preimplantation embryo undergoes DNA methylation reprogramming at the genome level by the processes of demethylation and methylation reestablishment.[24] Paternal genomes undergo demethylation in the hours following fertilization, immediately before DNA replication.[25],[26] In 2001, Santos et al.[27] investigated DNA methylation using immunofluorescence and established the first methylation reprogramming map in mouse preimplantation embryos. They found that DNA methylation was diminished step by step until the morula stage, and de novo methylation was observed in the inner cell mass (ICM).[27] The labeling of 5mC is very high in the 4-cell human embryo and declines thereafter. Unlike mouse embryos, remethylation can be detected in some nuclei of human embryos at the late morula stage. In addition, the labeling of 5mC is weaker in the ICM than it is in the trophectoderm.[28] Although we could, to some extent, investigate the DNA methylation profile using immunofluorescence, it is an indirect method and the interpretation of the results is largely dependent on subjective experience and thus subject to bias. Other techniques, such as methylation-specific polymerase chain reaction and bisulfite sequencing after replication, are options for assessing methylation status.[29],[30] More recently, the development of next-generation sequencing, especially single cell sequencing, has allowed researchers to more easily and precisely screen the whole methylome of gametes and embryos. Reduced representation bisulfite sequencing (RRBS) is a high-throughput method used to analyze genome-wide methylation profiles on a single nucleotide level with high base-pair resolution and enrichment for the CpG-dense regions.[31],[32] Smith et al.[23] used RRBS to study the DNA methylation pattern in mouse oocytes, spermatozoa, and embryos from zygotes through the postimplantation stage. They used hundreds of oocytes and embryos to provide a genome-scale base resolution of the DNA methylation profile.[23] In 2013, single-cell RRBS was developed to analyze the methylation profile at a single-base pair level within an individual cell.[33] Using this method can somewhat overcome the quantity limitation of human embryos and systematically dissect the DNA methylation map during the development of human preimplantation embryos. The major wave of global demethylation is completed at the 2-cell stage in the human embryo, which is different than it is in the mouse embryo. The demethylation process of the paternal genome is much faster than that of the maternal genome. In addition, in zygotes, the methylation level of male pronuclei is lower than that of female pronuclei. Another wave of demethylation occurs during the transition from morula to blastocyst. After implantation, a sharp increase in methylation occurs in the embryos. Compared to older elements, evolutionarily younger elements (long interspersed nuclear elements and short interspersed nuclear elements) show a milder demethylation process.[34],[35],[36] Currently, whole-genome bisulfite sequencing (WGBS) is the gold standard for detecting DNA methylation profiles, with >90% coverage in human genome.[37] WGBS generally requires microgram quantities of DNA and has limited applications in single-cell research. Post-bisulfite adaptor tagging is an alternative method, which requires only subnanogram quantities of DNA for WGBS of the whole genome.[38] The postbisulfite adaptor tagging method allows the assessment of 5mC heterogeneity in a single cell.[39] Tagmentation-based WGBS is an advanced method that can theoretically cover the complete methylome with a small quantity of cells.[40] A recent study explored the DNA methylation pattern of monkey embryos at the whole-genome level using a modified tagmentation-based WGBS method. Similar to human embryos, monkey embryos show genome demethylation and methylation reestablishment during preimplantation embryonic development. At the 8-cell stage, de novo methylation is more pronounced than demethylation. The similarity of the DNA methylation reprogramming process between humans and monkeys indicates that DNA remethylation in embryos is a highly conserved process among primates.[41]

Most DNA methylations have been found in CpG dinucleotides. However, current evidence indicates that sequences other than CpG, such as CpA, CpT, and CpC, may also be methylated.[42],[43] Ziller et al.[43] found that non-CpG methylation exists mainly in pluripotent cells, and decreases as the cells differentiate. In the somatic cells, non-CpG methylation is nearly nonexistent.[43] Nearly two-thirds of the methylcytosines in mouse germinal vesicle oocytes (GVOs) exist in non-CpG sites. As is the case with CpG methylation, non-CpG methylation shows significant enrichment in the gene bodies. GVOs have higher non-CpG methylation levels than nongrowing oocytes, indicating that this kind of DNA methylation accumulates along with the oocyte growth.[44]

 Histone Modification in Embryonic Development

Histones are the major components of chromatin and have an important impact on gene expression. In eukaryotes, the nucleosome is the basic unit of DNA packaging (chromatin).[45] Two of the four core histones (H2A, H2B, H3, and H4) make up a histone octamer. The N-terminal tails of histones can be modified, and the modification includes methylation and acetylation.[46] It has been reported that histone modification can regulate the establishment of the DNA methylation profile in the early embryo.[47] The interaction between Dnmt3L and H3 can be inhibited by methylation at lysine 4 of histone H3 (H3K4me), and as a consequence, de novo methylation at CpG islands may be prevented.[48] During the first cell cycle of mouse embryo, immunofluorescent staining showed differential expression of H3K9me and H3K27me between male and female pronuclei, which may explain the differences in the DNA demethylation processes between paternal and maternal genomes.[49] H3 methylation is also associated with cell fate and potency in mouse embryos. At the mouse 4-cell stage, blastomeres with the highest level of H3 methylation at specific arginine residues developed into ICM or polar trophectoderm cells.[50] In human embryonic stem cells (hESCs), ICM, cleavage-stage embryos, and gametes, histone H3 lysine 27 trimethylation (H3K27me3) regions show lower levels of DNA methylation, while there are much higher DNA methylation levels in regions free of H3K27me3 peaks. Interestingly, in human gametes, histone H3 lysine 9 trimethylation (H3K9me3) levels show a trend similar to that of DNA methylation. Active genes with the histone H3 lysine 4 trimethylation (H3K4me3) mark at the promoters in pluripotent hESCs are devoid of DNA methylation in both mature gametes and preimplantation embryos.[34],[51],[52],[53] Recently, a series of studies have provided a map of H3K4me3 and H3K27me3 modifications at the genome level in mouse gametes and preimplantation embryos. In mature oocytes, H3K4me3 has broad peaks at promoter regions and massive distal loci; the broad peaks are associated with partially methylated DNA domains. Knockdown of H3K4me3 by overexpressing KDM5B resulted in abnormal genome silencing in oocytes. After fertilization, the H3K4me3 peaks are diminished, and these peaks reappear following ZGA. Moreover, H3K4me3 is reestablished more quickly than H3K27me3, especially at the promoter regions.[54] Researchers found that there was a correlation between a broad H3K4me3 domain, high transcription activity, and cell identity, both in preimplantation development and in hESCs.[55] Microscale chromatin immunoprecipitation and sequencing showed that about 22% of the oocyte genome was related to broad H3K4me3. At the 2-cell stage, when ZGA begins, H3K4me3 becomes confined to the transcriptional start site regions.[56] These studies reveal the dynamic pattern of two important forms of histone modification, H3K4me3 and H3K27me3, during the early development of mouse embryos.

 Epigenetics and Assisted Reproductive Technology

Increasing evidences showed that children conceived through assisted reproductive technology (ART) procedures had higher risks of congenital birth defects, low birth weight (even singleton), and placental dysfunction.[57],[58],[59],[60],[61] The ART procedures affect the epigenomic profiles of gametes and early embryos, which may cause imprinting disorders.[22] Imprinting disorders are mainly caused by the alterations of the expression of multiple imprinted growth regulatory genes.[62] For example, abnormal expression of a paternal or maternal allele within the SNRPN-imprinted domain can lead to Angelman syndrome or Prader-Willi syndrome. In addition, aberration in the H19/IGF2 or KCNQ1 domains results in Beckwith–Wiedemann syndrome or Russell-Silver syndrome.[63] Since drastic methylation reprogramming occurs during germ cell and early embryonic development, preimplantation embryos are highly sensitive to imprinting defects.[64] In animal models, in vitro embryo culture and related procedures may result in imprinting disorders and impaired intrauterine growth.[65] Furthermore, vitrification of mouse embryos can alter the expression and methylation of H19 and Igf2 imprinted genes.[66] In both mice and humans, superovulated oocytes show altered DNA methylation profiles of the imprinted loci.[67] Similarly, in 32 of 50 (64%) of human early embryos produced by ART, the H19 differentially methylated region showed aberrant methylation patterns.[68] Nevertheless, all these studies focused on specific genes, the impact of ART procedures on the whole epigenome remains to be further investigated.


Epigenetic regulation, including DNA methylation and histone modification, plays an important role in the development of preimplantation embryos. Genome-wide epigenetic reprogramming during preimplantation embryonic development is a precise process that requires a dynamic balance. Aberrant regulation of this process may disrupt embryonic development or lead to clinical diseases such as imprinting disorders. High-throughput sequencing techniques allow us to investigate DNA methylation and histone modification at the genome-wide and single-base levels. However, although we have probed the epigenetic profile, our understanding of epigenetics is still far from complete. The practical coverage provided by the mainstream methods for detecting the whole epigenome remains relatively low. Moreover, the workflows associated with the methods are complex, making it difficult to increase the throughput. However, with the rapid improvement of next-generation sequencing techniques, we will have a comprehensive understanding of the whole epigenome in the preimplantation human embryo in the near future. Given the essential function of DNA methylation and histone modification in human embryonic development, such studies may suggest new markers that could complement morphological observation for embryonic selection in ART.

Financial support and sponsorship

This study was supported by the National High Technology Research and Development Program (grant number 2015AA020407) and the National Natural Science Foundation of China (grant numbers 81521002 and 31522034).

Conflicts of interest

There are no conflicts of interest.


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