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.

• MeSH
• Arvicolinae metabolism   MeSH
• DNA Methyltransferase 3A MeSH
• DNA (Cytosine-5-)-Methyltransferases * genetics   metabolism   MeSH
• Gerbillinae metabolism   MeSH
• Cricetinae MeSH
• Rats MeSH
• DNA Methylation * MeSH
• Guinea Pigs MeSH
• Muridae * metabolism   MeSH
• Mice MeSH
• Oocytes MeSH
• Transcription Factors metabolism   MeSH
• Animals MeSH
• Check Tag
• Cricetinae MeSH
• Rats MeSH
• Guinea Pigs MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA Methyltransferase 3A MeSH
• DNA (Cytosine-5-)-Methyltransferases * MeSH
• DNMT3L protein, human MeSH Browser
• Transcription Factors MeSH

BACKGROUND: Genes, principal units of genetic information, vary in complexity and evolutionary history. Less-complex genes (e.g., long non-coding RNA (lncRNA) expressing genes) readily emerge de novo from non-genic sequences and have high evolutionary turnover. Genesis of a gene may be facilitated by adoption of functional genic sequences from retrotransposon insertions. However, protein-coding sequences in extant genomes rarely lack any connection to an ancestral protein-coding sequence. RESULTS: We describe remarkable evolution of the murine gene D6Ertd527e and its orthologs in the rodent Muroidea superfamily. The D6Ertd527e emerged in a common ancestor of mice and hamsters most likely as a lncRNA-expressing gene. A major contributing factor was a long terminal repeat (LTR) retrotransposon insertion carrying an oocyte-specific promoter and a 5' terminal exon of the gene. The gene survived as an oocyte-specific lncRNA in several extant rodents while in some others the gene or its expression were lost. In the ancestral lineage of Mus musculus, the gene acquired protein-coding capacity where the bulk of the coding sequence formed through CAG (AGC) trinucleotide repeat expansion and duplications. These events generated a cytoplasmic serine-rich maternal protein. Knock-out of D6Ertd527e in mice has a small but detectable effect on fertility and the maternal transcriptome. CONCLUSIONS: While this evolving gene is not showing a clear function in laboratory mice, its documented evolutionary history in Muroidea during the last ~ 40 million years provides a textbook example of how a several common mutation events can support de novo gene formation, evolution of protein-coding capacity, as well as gene's demise.

• Keywords
• CAG, D6Ertd527e, De novo, Evolution, Gene, LTR, Oocyte, Polyserine, Retrotransposon,
• MeSH
• Muridae * MeSH
• RNA, Long Noncoding * genetics   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• RNA, Long Noncoding * MeSH

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.

• Keywords
• 5% oxygen, DNA methylation, In vitro culture, Mouse, Normoxia, Oocyte, Transcription,
• MeSH
• In Vitro Oocyte Maturation Techniques methods   MeSH
• Oxygen metabolism   MeSH
• DNA Methylation * MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Oocytes growth & development   MeSH
• Transcriptome * MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Oxygen MeSH

Meiotic maturation of oocyte relies on pre-synthesised maternal mRNA, the translation of which is highly coordinated in space and time. Here, we provide a detailed polysome profiling protocol that demonstrates a combination of the sucrose gradient ultracentrifugation in small SW55Ti tubes with the qRT-PCR-based quantification of 18S and 28S rRNAs in fractionated polysome profile. This newly optimised method, named Scarce Sample Polysome Profiling (SSP-profiling), is suitable for both scarce and conventional sample sizes and is compatible with downstream RNA-seq to identify polysome associated transcripts. Utilising SSP-profiling we have assayed the translatome of mouse oocytes at the onset of nuclear envelope breakdown (NEBD)-a developmental point, the study of which is important for furthering our understanding of the molecular mechanisms leading to oocyte aneuploidy. Our analyses identified 1847 transcripts with moderate to strong polysome occupancy, including abundantly represented mRNAs encoding mitochondrial and ribosomal proteins, proteasomal components, glycolytic and amino acids synthetic enzymes, proteins involved in cytoskeleton organization plus RNA-binding and translation initiation factors. In addition to transcripts encoding known players of meiotic progression, we also identified several mRNAs encoding proteins of unknown function. Polysome profiles generated using SSP-profiling were more than comparable to those developed using existing conventional approaches, being demonstrably superior in their resolution, reproducibility, versatility, speed of derivation and downstream protocol applicability.

• Keywords
• RNA-seq, SW55Ti rotor, mouse early embryo, mouse oocyte, mouse zygote, polysome fractionation, polysome profiling, translatome,
• MeSH
• Nuclear Envelope genetics   metabolism   MeSH
• Meiosis genetics   MeSH
• Mice MeSH
• Oocytes growth & development   metabolism   MeSH
• Polyribosomes genetics   MeSH
• RNA-Binding Proteins genetics   MeSH
• RNA, Messenger, Stored genetics   MeSH
• RNA, Ribosomal, 18S genetics   MeSH
• RNA, Ribosomal, 28S genetics   MeSH
• RNA-Seq MeSH
• Gene Expression Regulation, Developmental genetics   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• RNA-Binding Proteins MeSH
• RNA, Messenger, Stored MeSH
• RNA, Ribosomal, 18S MeSH
• RNA, Ribosomal, 28S MeSH

BACKGROUND: SIRT1 histone deacetylase acts on many epigenetic and non-epigenetic targets. It is thought that SIRT1 is involved in oocyte maturation; therefore, the importance of the ooplasmic SIRT1 pool for the further fate of mature oocytes has been strongly suggested. We hypothesised that SIRT1 plays the role of a signalling molecule in mature oocytes through selected epigenetic and non-epigenetic regulation. RESULTS: We observed SIRT1 re-localisation in mature oocytes and its association with spindle microtubules. In mature oocytes, SIRT1 distribution shows a spindle-like pattern, and spindle-specific SIRT1 action decreases α-tubulin acetylation. Based on the observation of the histone code in immature and mature oocytes, we suggest that SIRT1 is mostly predestined for an epigenetic mode of action in the germinal vesicles (GVs) of immature oocytes. Accordingly, BML-278-driven trimethylation of lysine K9 in histone H3 in mature oocytes is considered to be a result of GV epigenetic transformation. CONCLUSIONS: Taken together, our observations point out the dual spatiotemporal SIRT1 action in oocytes, which can be readily switched from the epigenetic to non-epigenetic mode of action depending on the progress of meiosis.

• Keywords
• Epigenetics, Histone code, In vitro maturation, Oocyte, SIRT1, Sirtuin 1,
• Publication type
• Journal Article MeSH