The enigma of DNA methylation in the mammalian oocyte.

The mammalian genome experiences profound setting and resetting of epigenetic patterns during the life-course. This is understood best for DNA methylation: the specification of germ cells, gametogenesis, and early embryo development are characterised by phases of widespread erasure and rewriting of methylation. While mitigating against intergenerational transmission of epigenetic information, these processes must also ensure correct genomic imprinting that depends on faithful and long-term memory of gamete-derived methylation states in the next generation. This underscores the importance of understanding the mechanisms of methylation programming in the germline. De novo methylation in the oocyte is of particular interest because of its intimate association with transcription, which results in a bimodal methylome unique amongst mammalian cells. Moreover, this methylation landscape is entirely set up in a non-dividing cell, making the oocyte a fascinating model system in which to explore mechanistic determinants of methylation. Here, we summarise current knowledge on the oocyte DNA methylome and how it is established, focussing on recent insights from knockout models in the mouse that explore the interplay between methylation and chromatin states. We also highlight some remaining paradoxes and enigmas, in particular the involvement of non-nuclear factors for correct de novo methylation

Effects of preovulatory aging on the developmental competence of mouse oocytes

Preovulatory aging of oocytes is caused by a delay in ovulation and it is known to impair postimplantation embryonic development. However, hardly anything is known about the molecular mechanisms associated with preovulatory aging. To investigate several aspects of RNA dynamics in the preovulatory-aged oocytes and possible consequences on early preimplantation development, the present study used a mouse model in which ovulation was postponed with the GnRH antagonist cetrorelix. Preovulatory aging led to a lower number of ovulated oocytes. Furthermore, preovulatory-aged oocytes were more difficult to fertilize, as was demonstrated by a decrease in 2-cell embryo rate after mating compared to controls. These results are in accordance with previous studies on preovulatory aging in several vertebrate models including rats and humans. As an indicator for transcriptional silencing and genome stability during oocyte growth, the epigenetic histone modification H3K9 trimethylation was analyzed using immunofluorescence. No effect of preovulatory aging on H3K9 trimethylation levels was observed. Before transcriptional silencing, mRNAs are stored in the oocyte until recruitment for protein translation later during oocyte maturation. One major key player in the storage and recruitment of maternal transcripts is the RNA-binding protein Ybx2. Immunofluorescent staining showed a significant decrease in Ybx2 protein abundance after preovulatory aging, which might have severe implications for RNA dynamics in the oocyte. Since the RNA-storage potential seemed impaired, transcript levels of selected maternal effect genes were analyzed by qRT-PCR. Maternal effect genes are of crucial importance for oocyte developmental potential because they function in the early embryo before transcription initiates again. Transcript levels of 2 out of 10 investigated ME genes (Smarca4 and Tet3) decreased significantly after preovulatory aging. Additionally, oligo(dT) priming allowed investigation of poly(A) tail length of mRNAs of the candidate maternal effect genes by qRT-PCR. The poly(A) tail determines the translation efficiency of transcripts in the oocyte and early embryo. The majority of genes showed elongation of poly(A) tail length after preovulatory aging. Similar results were also found after in vitro preovulatory aging of oocytes grown in follicle culture, although not the same genes were affected than in vivo. Loss of Ybx2 protein and polyadenylation of maternal effect gene transcripts should occur later during oocyte maturation. Therefore, the results imply premature recruitment of mRNAs for protein translation. This assumption is supported by experiments determining the start of transcription at the 2-cell stage of embryonic preimplantation development in the course of embryonic genome activation. Embryos were incubated in BrUTP, which intercalates into nascent RNA and can by visualized by immunofluorescence. Compared to controls, in embryos derived from preovulatory-aged oocytes an increase in transcription was observed, indicating a precocious start of embryonic genome activation. Last, DNA methylation maintenance of three imprinted genes (H19, Snrpn, Igf2r) during epigenetic reprogramming in the preimplantation embryo was analyzed. Deep amplicon bisulfite sequencing of single 8-cell embryos showed stable maintenance of DNA methylation at the investigated loci. Overall, the results show that the investigated processes are distinctly regulated in the oocyte and early embryo and not all of them are susceptible to preovulatory aging. Nevertheless, preovulatory aging impairs oocyte quality with possible long-term effects on embryonic development and reproductive success.Eine Verzögerung des Eisprungs führt zu präovulatorischer Überreife der Oozyte und dies kann die Entwicklung des Embryos nach der Einnistung in die Gebärmutter beeinträchtigen. Über die molekularen Mechanismen, die das Entwicklungspotenzial der überreifen Oozyte beeinflussen und die Auswirkungen auf die frühe Embryonalentwicklung ist nur wenig bekannt. Mit Hilfe eines Mausmodells, in dem der Eisprung mit Hilfe des GnRH Antagonisten Cetrorelix hinausgezögert wurde, wurden in dieser Studie verschiedene Aspekte der RNA Dynamik in der überreifen Oozyte und mögliche Konsequenzen für die frühe Embryonalentwicklung vor der Einnistung untersucht. Die Verzögerung des Eisprungs führte zu einer verringerten Anzahl von ovulierten, reifen Oozyten. Außerdem ließen sich überreife Oozyten schlechter befruchten, was zu einer Abnahme der Zahl von 2-Zell-Embryonen nach Verpaarung führte. Diese Ergebnisse stimmen mit vorhergegangenen Studien zur präovulatorischen Überreife in verschiedenen Vertebraten, u.a. Ratten und Menschen, überein. Während des Oozyten-Wachstums wird die Transkription eingestellt. Dieser Prozess wurde mittels Immunofluoreszenz gegen die epigenetische Histonmodifikation H3K9-Trimethylierung untersucht. Dabei wurde kein Effekt der präovulatorischen Überreife auf das Ausmaß der H3K9-Trimethylierung beobachtet. Vor dem transkriptionellen Arrest werden mRNAs in der Oozyte gespeichert, bis sie im späteren Verlauf der Oozyten-Reifung für die Translation von Proteinen rekrutiert werden. Ein wichtiger Faktor für die Speicherung und Rekrutierung von mRNA in der Oozyte ist das RNA-Bindeprotein Ybx2. Mittels Immunofluoreszenz konnte eine signifikante Abnahme der Ybx2 Proteinmenge nach präovulatorischer Überreife gezeigt werden. Dies könnte zu Störungen der RNA-Speicherung in der Oozyte führen. Um dies zu ermitteln wurde die Transkriptmenge von ausgewählten maternalen Effektgenen (ME-Genen) mittels qRT-PCR untersucht. Transkripte der ME-Gene sind entscheidend für das Entwicklungspotential der Oozyte, da sie im frühen Embryo, dessen Transkription noch nicht begonnen hat, wirken. Von zwei der 10 untersuchten ME-Gene (Smarca4 und Tet3) waren die Transkriptmengen, im Vergleich zu den Kontrollen, in überreifen Oozyten signifikant reduziert. Außerdem wurde die Poly(A)-Schwanzlänge von ME-Genen untersucht, da diese die Effizienz der Translation eines Transkriptes in der Oozyte bestimmt. In überreifen Oozyten zeigte der Großteil der analysierten Kandidatengene eine Verlängerung des Poly(A)-Schwanzes. Ähnliche Ergebnisse wurden auch bei in vitro präovulatorisch überreifen Oozyten aus einer Follikelkultur beobachtet, obwohl hier nicht die gleichen ME-Gene beeinträchtigt waren. Die beobachtete Abnahme von Ybx2-Protein und die Verlängerung des Poly(A)-Schwanzes der ME-Transkripte sind Prozesse, die während der Eizell-Reifung eigentlich später stattfinden. Die Beobachtung, dass diese Prozesse bei präovulatorischer Alterung früher stattfinden, impliziert, dass auch die Rekrutierung von mRNA für die Proteintranslation verfrüht stattfinden könnte, was die Transkription im Embryo beeinflussen kann. Diese Annahme wird durch Analysen zur Aktivierung der Transkription im 2-Zell-Embryo unterstützt. Embryonen wurden in BrUTP inkubiert, welches sich während der Transkription in die RNA einlagert und mit Immunofluoreszenz nachgewiesen werden kann. Dieses Experiment zeigte, im Vergleich zu Kontrollen, eine Zunahme der Transkription in Embryonen von überreifen Oozyten, was auf einen vorzeitigen Start der embryonalen Genomaktivierung hindeutet. Zuletzt wurde die DNA-Methylierung von drei Genen, die dem Imprinting unterliegen (H19, Snrpn, Igf2r), in einzelnen 8-Zell-Embryonen analysiert. Deep amplicon bisulifte sequencing zeigte einen stabilen Erhalt der DNA-Methylierung nach präovulatorischer Überreife in den untersuchten Loci. Insgesamt zeigen diese Ergebnisse, dass die untersuchten Prozesse in der Oozyte und im frühen Embryo individuell reguliert sind und nicht alle durch präovulatorische Überreife gestört werden. Dennoch lässt sich sagen, dass präovulatorische Überreife die Qualität der Oozyte beeinträchtigt, mit möglichen Langzeiteffekten auf die Embryo-Entwicklung und den Reproduktionserfolg

Epigenetic regulation in development: is the mouse a good model for the human?

BACKGROUND: Over the past few years, advances in molecular technologies have allowed unprecedented mapping of epigenetic modifications in gametes and during early embryonic development. This work is allowing a detailed genomic analysis, which for the first time can answer long-standing questions about epigenetic regulation and reprogramming, and highlights differences between mouse and human, the implications of which are only beginning to be explored. OBJECTIVE AND RATIONALE: In this review, we summarise new low-cell molecular methods enabling the interrogation of epigenetic information in gametes and early embryos, the mechanistic insights these have provided, and contrast the findings in mouse and human. SEARCH METHODS: Relevant studies were identified by PubMed search. OUTCOMES: We discuss the levels of epigenetic regulation, from DNA modifications to chromatin organisation, during mouse gametogenesis, fertilisation and pre- and post-implantation development. The recently characterised features of the oocyte epigenome highlight its exceptionally unique regulatory landscape. The chromatin organisation and epigenetic landscape of both gametic genomes are rapidly reprogrammed after fertilisation. This extensive epigenetic remodelling is necessary for zygotic genome activation, but the mechanistic link remains unclear. While the vast majority of epigenetic information from the gametes is erased in pre-implantation development, new insights suggest that repressive histone modifications from the oocyte may mediate a novel mechanism of imprinting. To date, the characterisation of epigenetics in human development has been almost exclusively limited to DNA methylation profiling; these data reinforce that the global dynamics are conserved between mouse and human. However, as we look closer, it is becoming apparent that the mechanisms regulating these dynamics are distinct. These early findings emphasise the importance of investigations of fundamental epigenetic mechanisms in both mouse and humans. WIDER IMPLICATIONS: Failures in epigenetic regulation have been implicated in human disease and infertility. With increasing maternal age and use of reproductive technologies in countries all over the world, it is becoming ever more important to understand the necessary processes required to establish a developmentally competent embryo. Furthermore, it is essential to evaluate the extent to which these epigenetic patterns are sensitive to such technologies and other adverse environmental exposures

DNA Methylation Dynamics in the Female Germline and Maternal-Effect Mutations That Disrupt Genomic Imprinting.

Genomic imprinting is an epigenetic marking process that results in the monoallelic expression of a subset of genes. Many of these 'imprinted' genes in mice and humans are involved in embryonic and extraembryonic growth and development, and some have life-long impacts on metabolism. During mammalian development, the genome undergoes waves of (re)programming of DNA methylation and other epigenetic marks. Disturbances in these events can cause imprinting disorders and compromise development. Multi-locus imprinting disturbance (MLID) is a condition by which imprinting defects touch more than one locus. Although most cases with MLID present with clinical features characteristic of one imprinting disorder. Imprinting defects also occur in 'molar' pregnancies-which are characterized by highly compromised embryonic development-and in other forms of reproductive compromise presenting clinically as infertility or early pregnancy loss. Pathogenic variants in some of the genes encoding proteins of the subcortical maternal complex (SCMC), a multi-protein complex in the mammalian oocyte, are responsible for a rare subgroup of moles, biparental complete hydatidiform mole (BiCHM), and other adverse reproductive outcomes which have been associated with altered imprinting status of the oocyte, embryo and/or placenta. The finding that defects in a cytoplasmic protein complex could have severe impacts on genomic methylation at critical times in gamete or early embryo development has wider implications beyond these relatively rare disorders. It signifies a potential for adverse maternal physiology, nutrition, or assisted reproduction to cause epigenetic defects at imprinted or other genes. Here, we review key milestones in DNA methylation patterning in the female germline and the embryo focusing on humans. We provide an overview of recent findings regarding DNA methylation deficits causing BiCHM, MLID, and early embryonic arrest. We also summarize identified SCMC mutations with regard to early embryonic arrest, BiCHM, and MLID

Increased transcriptome variation and localised DNA methylation changes in oocytes from aged mice revealed by parallel single-cell analysis.

Advancing maternal age causes a progressive reduction in fertility. The decline in developmental competence of the oocyte with age is likely to be a consequence of multiple contributory factors. Loss of epigenetic quality of the oocyte could impair early developmental events or programme adverse outcomes in offspring that manifest only later in life. Here, we undertake joint profiling of the transcriptome and DNA methylome of individual oocytes from reproductively young and old mice undergoing natural ovulation. We find reduced complexity as well as increased variance in the transcriptome of oocytes from aged females. This transcriptome heterogeneity is reflected in the identification of discrete sub-populations. Oocytes with a transcriptome characteristic of immature chromatin configuration (NSN) clustered into two groups: one with reduced developmental competence, as indicated by lower expression of maternal effect genes, and one with a young-like transcriptome. Oocytes from older females had on average reduced CpG methylation, but the characteristic bimodal methylation landscape of the oocyte was preserved. Germline differentially methylated regions of imprinted genes were appropriately methylated irrespective of age. For the majority of differentially expressed transcripts, the absence of correlated methylation changes suggests a post-transcriptional basis for most age-related effects on the transcriptome. However, we did find differences in gene body methylation at which there were corresponding changes in gene expression, indicating age-related effects on transcription that translate into methylation differences. Interestingly, oocytes varied in expression and methylation of these genes, which could contribute to variable competence of oocytes or penetrance of maternal age-related phenotypes in offspring

Landscape of Genome-Wide DNA Methylation of Colorectal Cancer Metastasis.

Colorectal cancer is a heterogeneous disease caused by both genetic and epigenetics factors. Analysing DNA methylation changes occurring during colorectal cancer progression and metastasis formation is crucial for the identification of novel epigenetic markers of patient prognosis. Genome-wide methylation sequencing of paired samples of colon (normal adjacent, primary tumour and lymph node metastasis) showed global hypomethylation and CpG island (CGI) hypermethylation of primary tumours compared to normal. In metastasis we observed high global and non-CGI regions methylation, but lower CGI methylation, compared to primary tumours. Gene ontology analysis showed shared biological processes between hypermethylated CGIs in metastasis and primary tumours. After complementary analysis with The Cancer Genome Atlas (TCGA) cohort, FIGN, HTRA3, BDNF, HCN4 and STAC2 genes were found associated with poor survival. We mapped the methylation landscape of colon normal tissues, primary tumours and lymph node metastasis, being capable of identified methylation changes throughout the genome. Furthermore, we found five genes with potential for methylation biomarkers of poor prognosis in colorectal cancer patients

A KHDC3L mutation resulting in recurrent hydatidiform mole causes genome-wide DNA methylation loss in oocytes and persistent imprinting defects post-fertilisation

Abstract: Background: Maternal effect mutations in the components of the subcortical maternal complex (SCMC) of the human oocyte can cause early embryonic failure, gestational abnormalities and recurrent pregnancy loss. Enigmatically, they are also associated with DNA methylation abnormalities at imprinted genes in conceptuses: in the devastating gestational abnormality biparental complete hydatidiform mole (BiCHM) or in multi-locus imprinting disease (MLID). However, the developmental timing, genomic extent and mechanistic basis of these imprinting defects are unknown. The rarity of these disorders and the possibility that methylation defects originate in oocytes have made these questions very challenging to address. Methods: Single-cell bisulphite sequencing (scBS-seq) was used to assess methylation in oocytes from a patient with BiCHM identified to be homozygous for an inactivating mutation in the human SCMC component KHDC3L. Genome-wide methylation analysis of a preimplantation embryo and molar tissue from the same patient was also performed. Results: High-coverage scBS-seq libraries were obtained from five KHDC3Lc.1A>G oocytes, which revealed a genome-wide deficit of DNA methylation compared with normal human oocytes. Importantly, germline differentially methylated regions (gDMRs) of imprinted genes were affected similarly to other sequence features that normally become methylated in oocytes, indicating no selectivity towards imprinted genes. A range of methylation losses was observed across genomic features, including gDMRs, indicating variable sensitivity to defects in the SCMC. Genome-wide analysis of a pre-implantation embryo and molar tissue from the same patient showed that following fertilisation methylation defects at imprinted genes persist, while most non-imprinted regions of the genome recover near-normal methylation post-implantation. Conclusions: We show for the first time that the integrity of the SCMC is essential for de novo methylation in the female germline. These findings have important implications for understanding the role of the SCMC in DNA methylation and for the origin of imprinting defects, for counselling affected families, and will help inform future therapeutic approaches

Novel Methylation Biomarkers for Colorectal Cancer Prognosis

Colorectal cancer (CRC) comprises the third most common cancer worldwide and the second regarding number of deaths. In order to make a correct and early diagnosis to predict metastasis formation, biomarkers are an important tool. Although there are multiple signaling pathways associated with cancer progression, the most recognized are the MAPK pathway, p53 pathway, and TGF-β pathway. These pathways regulate many important functions in the cell, such as cell cycle regulation, proliferation, differentiation, and metastasis formation, among others. Changes in expression in genes belonging to these pathways are drivers of carcinogenesis. Often these expression changes are caused by mutations; however, epigenetic changes, such as DNA methylation, are increasingly acknowledged to play a role in the deregulation of oncogenic genes. This makes DNA methylation changes an interesting biomarkers in cancer. Among the newly identified biomarkers for CRC metastasis INHBB, SMOC2, BDNF, and TBRG4 are included, all of which are highly deregulated by methylation and closely associated with metastasis. The identification of such biomarkers in metastasis of CRC may allow a better treatment and early identification of cancer formation in order to perform better diagnostics and improve the life expectancy

Preovulatory Aging In Vivo and In Vitro Affects Maturation Rates, Abundance of Selected Proteins, Histone Methylation Pattern and Spindle Integrity in Murine Oocytes

Demond H, Trapphoff T, Dankert D, et al. Preovulatory Aging In Vivo and In Vitro Affects Maturation Rates, Abundance of Selected Proteins, Histone Methylation Pattern and Spindle Integrity in Murine Oocytes. PLOS ONE. 2016;11(9): e0162722.Delayed ovulation and delayed fertilization can lead to reduced developmental competence of the oocyte. In contrast to the consequences of postovulatory aging of the oocyte, hardly anything is known about the molecular processes occurring during oocyte maturation if ovulation is delayed (preovulatory aging). We investigated several aspects of oocyte maturation in two models of preovulatory aging: an in vitro follicle culture and an in vivo mouse model in which ovulation was postponed using the GnRH antagonist cetrorelix. Both models showed significantly reduced oocyte maturation rates after aging. Furthermore, in vitro preovulatory aging deregulated the protein abundance of the maternal effect genes Smarca4 and Nlrp5, decreased the levels of histone H3K9 trimethylation and caused major deterioration of chromosome alignment and spindle conformation. Protein abundance of YBX2, an important regulator of mRNA stability, storage and recruitment in the oocyte, was not affected by in vitro aging. In contrast, in vivo preovulatory aging led to reduction in Ybx2 transcript and YBX2 protein abundance. Taken together, preovulatory aging seems to affect various processes in the oocyte, which could explain the low maturation rates and the previously described failures in fertilization and embryonic development