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Transcriptome and DNA methylation profiling during the NSN to SN transition in mouse oocytes [Bisulfite-Seq]

ABSTRACT: During oocyte maturation, the transition from a non-surrounded nucleolus (NSN) to a surrounded nucleolus (SN) stage coincides with dramatic chromatin reconfiguration and transcriptional silencing and is crucial for developmental competence. To study the transcriptome and DNA methylation dynamics during the NSN to SN transition we used single cell (sc)M&T-seq to generate scRNA-seq and sc bisulphite-seq data from GV oocytes, classified as NSN or SN by Hoechst staining of their nuclei. Transcriptome analysis showed a lower number of detected transcripts in SN oocytes as well as downregulation of 576 genes, which were enriched for processes related to mRNA processing. We used the RNA-seq data to generate a classifier, which can infer chromatin stage in scRNA-seq data sets, based on their transcription profile. The classifier was successfully tested in multiple published datasets of mouse models with a known skew in NSN:SN ratios. DNA methylation analysis showed increased methylation in SN compared to NSN oocytes, which was most pronounced in regions with intermediate levels of DNA methylation. Overlap with ChIP-seq data for the histone modifications H3K36me3, H3K4me3 and H3K27me3 showed that regions gaining methylation in SN oocytes are enriched for overlapping H3K36me3 and H3K27me3, which is unusual as these marks do not typically coincide. We believe that these late-methylating SN regions are in regions with high chromatin plasticity and that the overlap of H3K36me3 and H3K27me3 may indicate a transient switch or heterogeneity on a single-cell level.

ORGANISM(S): Mus musculus

PROVIDER: GSE274300 | GEO | 2025/01/02

REPOSITORIES: GEO

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Project description:Mammalian oogenesis is a complex mechanism entailing the maturation of primordial follicles into preovulatory follicles followed by the release of antral oocytes. In the ovary, the antral compartment consists of two populations of oocytes whose main difference regards the ability to resume meiosis and to develop, after the egg-sperm fusion, until the blastocyst stage. Still inexplicably, the antral oocytes distinguishable as Surrounded Nucleolus (SN; 70% of the antral oocytes population) are able to develop to the blastocyst stage, while those showing a Not Surrounded Nucleolus (NSN) arrest at the two-cell stage. RNA labeling and hybridization on microarray was done independently for SN and NSN oocytes with 2 replications. Two sets of 50 antral oocytes were collected in M2 medium and then were labelled with Cy3-CTP. Fluorescently labelled microarray targets were prepared using a Low RNA Input Fluorescent Linear Amplification Kit (Agilent) with 2-round amplification. Cy5-CTP-labeled reference target was produced from mixture of Stratagene Universal Mouse Reference RNA and MC1 cell RNA. Targets were purified using an RNeasy Mini Kit (Qiagen), and then quantified on a NanoDrop scanning spectrophotometer (NanoDrop Technologies). Target cRNA was hybridized to the NIA Mouse 44K Microarray v3.0 (whole genome 60-mer oligo arrays, Agilent Technology, design ID 015087) according to manufacturers protocol (Two-Color Microarray-Based Gene Expression Analysis Protocol, Product # G4140-90050, Version 5.0.1). Slides were scanned with an Agilent DNA Microarray Scanner (model G2505-64120) at 100% and 10% PMT in both channels (except for the two oocyte Samples which were only scanned once each), with a scan resolution of 5um. All hybridizations compared one Cy3-CTP-labeled experimental target to the single Cy5-CTP-labeled reference target which was used for normalization. In the final analysis, we made a cutoff of 2.0 for the processed data (i.e., if value<2.0, it is set as 2.0) to minimize the noises from 2-round linear amplifications.

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Transcriptome and DNA methylation profiling during the NSN to SN transition in mouse oocytes [Bisulfite-Seq]

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