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Highly sensitive sequencing reveals dynamic modifications and activities of small RNAs in mouse oocytes and early embryos

ABSTRACT: Small RNAs play important roles in early embryonic development. However, their expression dynamics and modifications are poorly understood because of the scarcity of RNA that is obtainable for sequencing analysis. Using an improved deep sequencing method that requires as little as 10 ng of total RNA or 50 oocytes, we profile small RNAs in mouse oocytes and early embryos. We find that microRNA (miRNA) expression starts soon after fertilization, and the mature miRNAs carried into the zygote by sperm during fertilization are relatively rare compared to the oocyte miRNAs. Intriguingly, the zygotic miRNAs display a marked increase in 3′ mono- and oligoadenylation in one- to two-cell embryos, which may protect the miRNAs from the massive degradation taking place during that time. Moreover, bioinformatics analyses show that the function of miRNA is suppressed from the oocyte to the two-cell stage and appears to be reactivated after the two-cell stage to regulate genes important in embryonic development. Our study thus provides a highly sensitive profiling method and valuable data sets for further examination of small RNAs in early embryos.

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE83581 | GEO | 2016/06/23

REPOSITORIES: GEO

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Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare small non-coding RNA profiling (snRNA-seq) in WT oocyte, sperm and 2PN stage embryos to those sperm and 2PN stage embryos derived from WT, Dicer cKO and Drosha cKO. We further study the roles of sperm-borne small RNA on fertilization and pre-implantation embryonic development. Methods: Small RNA profiles of adult wild-type (WT) oocytes, adult WT sperm, 2PN stage embryos, adult Dicer cKO/Drosha cKO sperm, 2PN stage embryos were generated by deep sequencing in duplicate, using Ion Torrent Proton. The sequence reads that passed quality filters were analyzed at the small RNA level with two methods: Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (build mm9) and identified 16,014 small RNA (miRNA and endo-siRNA) in the oocyte, sperm and 2PN stage of WT and Dicer cKO/Drosha cKO mice with BWA workflow and 34,115 transcripts with TopHat workflow. Approximately 47% of the miRNAs showed differential expression between the WT and Dicer cKO sperm, ~52% of miRNAs were shown dysregulated in Drosha cKO sperm compared to those in WT sperm with a fold change ≥2.0 and p value <0.05. Data analysis with BWA and TopHat workflows revealed a significant overlap yet provided complementary insights in transcriptome profiling. Conclusions: Our study represents the first detailed analysis of small non-coding RNAs (miRNAs) in sperm and demonstrated that sperm-borne small RNAs are important for fertilization and early embrynic develoment, with biologic duplicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of small RNAs profiles in mouse sperm, oocytes and 2PN stage of embryos. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of small RNA contents within sperm or oocytes/embryos. We conclude that RNA-seq based small RNAs characterization in gametes would expedite genetic network analyses and permit the dissection of complex biologic functions during fertilization and embryonic development.

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Highly sensitive sequencing reveals dynamic modifications and activities of small RNAs in mouse oocytes and early embryos

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