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KAS-seq profiling captures transcription dynamics during oocyte maturation

ABSTRACT: In fully grown oocytes, the genome is considered to be globally transcriptionally silenced. However, this conclusion is primarily derived from the results obtained through immunofluorescence staining or inferred from the highly condensed state of chromosomes, lacking more direct evidence. Here, by using a kethoxal-assisted single-stranded DNA sequencing (KAS-seq) approach, we investigated the landscape of single-strand DNA (ssDNA) throughout the genome and provided a readout of the activity and dynamics of transcription during oocyte meiotic maturation. In non-surrounded nucleolus (NSN) oocytes, we observed a robust KAS-seq signal, indicating the high transcriptional activity. In surrounded nucleolus (SN) oocytes, the presence of ssDNA still persists although the KAS-seq signal was relatively weak, suggesting the presence of transcription. Accompanying with the meiotic resumption, the transcriptional activity gradually decreased, and global repression was detected in matured oocytes. Moreover, we preformed the integrative genomics analysis to dissect the transcriptional dynamics during mouse oocyte maturation. In sum, the present study delineates the detailed transcriptional activity during mammalian oocyte maturation.

ORGANISM(S): Mus musculus

PROVIDER: GSE252350 | GEO | 2024/06/07

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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KAS-seq profiling captures transcription dynamics during oocyte maturation

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