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

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

Endogenous retroviral insertions drive non-canonical imprinting in extra-embryonic tissues.

BACKGROUND: Genomic imprinting is an epigenetic phenomenon that allows a subset of genes to be expressed mono-allelically based on the parent of origin and is typically regulated by differential DNA methylation inherited from gametes. Imprinting is pervasive in murine extra-embryonic lineages, and uniquely, the imprinting of several genes has been found to be conferred non-canonically through maternally inherited repressive histone modification H3K27me3. However, the underlying regulatory mechanisms of non-canonical imprinting in post-implantation development remain unexplored. RESULTS: We identify imprinted regions in post-implantation epiblast and extra-embryonic ectoderm (ExE) by assaying allelic histone modifications (H3K4me3, H3K36me3, H3K27me3), gene expression, and DNA methylation in reciprocal C57BL/6 and CAST hybrid embryos. We distinguish loci with DNA methylation-dependent (canonical) and independent (non-canonical) imprinting by assaying hybrid embryos with ablated maternally inherited DNA methylation. We find that non-canonical imprints are localized to endogenous retrovirus-K (ERVK) long terminal repeats (LTRs), which act as imprinted promoters specifically in extra-embryonic lineages. Transcribed ERVK LTRs are CpG-rich and located in close proximity to gene promoters, and imprinting status is determined by their epigenetic patterning in the oocyte. Finally, we show that oocyte-derived H3K27me3 associated with non-canonical imprints is not maintained beyond pre-implantation development at these elements and is replaced by secondary imprinted DNA methylation on the maternal allele in post-implantation ExE, while being completely silenced by bi-allelic DNA methylation in the epiblast. CONCLUSIONS: This study reveals distinct epigenetic mechanisms regulating non-canonical imprinted gene expression between embryonic and extra-embryonic development and identifies an integral role for ERVK LTR repetitive elements

Transcription and chromatin determinants of de novo DNA methylation timing in oocytes.

BACKGROUND: Gametogenesis in mammals entails profound re-patterning of the epigenome. In the female germline, DNA methylation is acquired late in oogenesis from an essentially unmethylated baseline and is established largely as a consequence of transcription events. Molecular and functional studies have shown that imprinted genes become methylated at different times during oocyte growth; however, little is known about the kinetics of methylation gain genome wide and the reasons for asynchrony in methylation at imprinted loci. RESULTS: Given the predominant role of transcription, we sought to investigate whether transcription timing is rate limiting for de novo methylation and determines the asynchrony of methylation events. Therefore, we generated genome-wide methylation and transcriptome maps of size-selected, growing oocytes to capture the onset and progression of methylation. We find that most sequence elements, including most classes of transposable elements, acquire methylation at similar rates overall. However, methylation of CpG islands (CGIs) is delayed compared with the genome average and there are reproducible differences amongst CGIs in onset of methylation. Although more highly transcribed genes acquire methylation earlier, the major transitions in the oocyte transcriptome occur well before the de novo methylation phase, indicating that transcription is generally not rate limiting in conferring permissiveness to DNA methylation. Instead, CGI methylation timing negatively correlates with enrichment for histone 3 lysine 4 (H3K4) methylation and dependence on the H3K4 demethylases KDM1A and KDM1B, implicating chromatin remodelling as a major determinant of methylation timing. We also identified differential enrichment of transcription factor binding motifs in CGIs acquiring methylation early or late in oocyte growth. By combining these parameters into multiple regression models, we were able to account for about a fifth of the variation in methylation timing of CGIs. Finally, we show that establishment of non-CpG methylation, which is prevalent in fully grown oocytes, and methylation over non-transcribed regions, are later events in oogenesis. CONCLUSIONS: These results do not support a major role for transcriptional transitions in the time of onset of DNA methylation in the oocyte, but suggest a model in which sequences least dependent on chromatin remodelling are the earliest to become permissive for methylation

Setd1b, encoding a histone 3 lysine 4 methyltransferase, is a maternal effect gene required for the oogenic gene expression program.

Germ cell development involves major reprogramming of the epigenome to prime the zygote for totipotency. Histone 3 lysine 4 (H3K4) methylations are universal epigenetic marks mediated in mammals by six H3K4 methyltransferases related to fly Trithorax, including two yeast Set1 orthologs: Setd1a and Setd1b. Whereas Setd1a plays no role in oogenesis, we report that Setd1b deficiency causes female sterility in mice. Oocyte-specific Gdf9-iCre conditional knockout (Setd1bGdf9 cKO) ovaries develop through all stages; however, follicular loss accumulated with age and unfertilized metaphase II (MII) oocytes exhibited irregularities of the zona pellucida and meiotic spindle. Most Setd1bGdf9 cKO zygotes remained in the pronuclear stage and displayed polyspermy in the perivitelline space. Expression profiling of Setd1bGdf9 cKO MII oocytes revealed (1) that Setd1b promotes the expression of the major oocyte transcription factors including Obox1, 2, 5, 7, Meis2 and Sall4; and (2) twice as many mRNAs were upregulated than downregulated, suggesting that Setd1b also promotes the expression of negative regulators of oocyte development with multiple Zfp-KRAB factors implicated. Together, these findings indicate that Setd1b serves as maternal effect gene through regulation of the oocyte gene expression program

Expression analysis suggests that DNMT3L is required for oocyte de novo DNA methylation only in Muridae and Cricetidae rodents

Abstract Background During early mammalian development, DNA methylation undergoes two waves of reprogramming, enabling transitions between somatic cells, oocyte and embryo. The first wave of de novo DNA methylation establishment occurs in oocytes. Its molecular mechanisms have been studied in mouse, a classical mammalian model. Current model describes DNA methyltransferase 3A (DNMT3A) and its cofactor DNMT3L as two essential factors for oocyte DNA methylation—the ablation of either leads to nearly complete abrogation of DNA methylation. However, DNMT3L is not expressed in human oocytes, suggesting that the mechanism uncovered in mouse is not universal across mammals. Results We analysed available RNA-seq data sets from oocytes of multiple mammals, including our novel data sets of several rodent species, and revealed that Dnmt3l is expressed only in the oocytes of mouse, rat and golden hamster, and at a low level in guinea pigs. We identified a specific promoter sequence recognised by an oocyte transcription factor complex associated with strong Dnmt3l activity and demonstrated that it emerged in the rodent clade Eumuroida, comprising the families Muridae (mice, rats, gerbils) and Cricetidae (hamsters). In addition, an evolutionarily novel promoter emerged in the guinea pig, driving weak Dnmt3l expression, likely without functional relevance. Therefore, Dnmt3l is expressed and consequently plays a role in oocyte de novo DNA methylation only in a small number of rodent species, instead of being an essential pan-mammalian factor. In contrast to somatic cells, where catalytically inactive DNMT3B interacts with DNMT3A, forming a heterotetramer, we did not find evidence for the expression of such inactive Dnmt3b isoforms in the oocytes of the tested species. Conclusions The analysis of RNA-seq data and genomic sequences revealed that DNMT3L is likely to play a role in oocytes de novo DNA methylation only in mice, rats, gerbils and hamsters. The mechanism governing de novo DNA methylation in the oocytes of most mammalian species, including humans, occurs through a yet unknown mechanism that differs from the current model discovered in mouse

Endogenous retroviral insertions drive non-canonical imprinting in extra-embryonic tissues.

BACKGROUND: Genomic imprinting is an epigenetic phenomenon that allows a subset of genes to be expressed mono-allelically based on the parent of origin and is typically regulated by differential DNA methylation inherited from gametes. Imprinting is pervasive in murine extra-embryonic lineages, and uniquely, the imprinting of several genes has been found to be conferred non-canonically through maternally inherited repressive histone modification H3K27me3. However, the underlying regulatory mechanisms of non-canonical imprinting in post-implantation development remain unexplored. RESULTS: We identify imprinted regions in post-implantation epiblast and extra-embryonic ectoderm (ExE) by assaying allelic histone modifications (H3K4me3, H3K36me3, H3K27me3), gene expression, and DNA methylation in reciprocal C57BL/6 and CAST hybrid embryos. We distinguish loci with DNA methylation-dependent (canonical) and independent (non-canonical) imprinting by assaying hybrid embryos with ablated maternally inherited DNA methylation. We find that non-canonical imprints are localized to endogenous retrovirus-K (ERVK) long terminal repeats (LTRs), which act as imprinted promoters specifically in extra-embryonic lineages. Transcribed ERVK LTRs are CpG-rich and located in close proximity to gene promoters, and imprinting status is determined by their epigenetic patterning in the oocyte. Finally, we show that oocyte-derived H3K27me3 associated with non-canonical imprints is not maintained beyond pre-implantation development at these elements and is replaced by secondary imprinted DNA methylation on the maternal allele in post-implantation ExE, while being completely silenced by bi-allelic DNA methylation in the epiblast. CONCLUSIONS: This study reveals distinct epigenetic mechanisms regulating non-canonical imprinted gene expression between embryonic and extra-embryonic development and identifies an integral role for ERVK LTR repetitive elements

Increased Expression of Maturation Promoting Factor Components Speeds Up Meiosis in Oocytes from Aged Females

The rate of chromosome segregation errors that emerge during meiosis I in the mammalian female germ line are known to increase with maternal age; however, little is known about the underlying molecular mechanism. The objective of this study was to analyze meiotic progression of mouse oocytes in relation to maternal age. Using the mouse as a model system, we analyzed the timing of nuclear envelope breakdown and the morphology of the nuclear lamina of oocytes obtained from young (2 months old) and aged females (12 months old). Oocytes obtained from older females display a significantly faster progression through meiosis I compared to the ones obtained from younger females. Furthermore, in oocytes from aged females, lamin A/C structures exhibit rapid phosphorylation and dissociation. Additionally, we also found an increased abundance of MPF components and increased translation of factors controlling translational activity in the oocytes of aged females. In conclusion, the elevated MPF activity observed in aged female oocytes affects precocious meiotic processes that can multifactorially contribute to chromosomal errors in meiosis I

MOESM9 of Transcription and chromatin determinants of de novo DNA methylation timing in oocytes

Additional file 9: Table S8. Expression levels of transcription factors with binding motifs enriched in CGIs with low methylation level in 60–65 µm oocytes

MOESM5 of Transcription and chromatin determinants of de novo DNA methylation timing in oocytes

Additional file 5: Table S4. Sequencing statistics for ssRNA-seq libraries from NGO, size-selected and GV oocytes