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First cleavage is a manifestation of the geometry of the unfertilized oocyte: implications for monozygotic twinning in mice.

ABSTRACT: A long-standing question in mammalian embryology is whether regional differences of oocyte composition matter for the properties of blastomeres receiving those regions after fertilization. A hitherto untested hypothesis is that allocation depends on the orientation of 1st cleavage. However, the orientation is influenced by the site of sperm entry in the oocyte, which can be almost anywhere on the membrane of oocytes when these are inseminated. Therefore, we harnessed the intracytoplasmic sperm injection to impose the site of fertilization in three specific ooplasmic regions (animal pole, vegetal pole, equator) of mouse oocytes. Notwithstanding this categorical distinction, the sister blastomeres of resultant 2-cell embryos differed from each other in the same way, as measured by gene expression and twin blastocysts formation following bisection. We reasoned that either the oocyte territories did not matter, or their effect was obscured by other factors. To shed light on these possibilities, we immobilized the oocytes on the micromanipulation stage during sperm injection and for 24 h thereafter. Imaging revealed that the orientation of 1st cleavage, instead of depending on the fertilization site, depended on the shorter diameter of the unfertilized oocyte. This even led to the segregation of animal and vegetal hemispheres in the blastomeres of 2-cell embryos. We conclude that 1stcleavage is a manifestation of the intrinsic organization of the oocyte prior to fertilization, and that oocyte geometry factors in to the patterning of mouse embryos more so than fertilization topology.

ORGANISM(S): Mus musculus

PROVIDER: GSE241089 | GEO | 2024/03/01

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.

Project description:RNA transcripts are distributed non-uniformly in the oocytes of many animals, such that newly-divided embryo cells (blastomeres) inherit distinct transcriptomes following fertilization. In animals such as the frog, Xenopus laevis, programmed transcript regionalization directs early embryonic axis formation and is essential for normal development. However, it is unknown whether such transcriptome asymmetry directs embryogenesis in mammals, or indeed, whether it occurs at all. We here address this by transcript profiling of matching sub-cellular structures and single cells in mouse oocytes and early embryos and analytical strategies exploiting the paired data structure. Spindle samples contained a set of transcripts that was distinguishable from that of the unfertilized metaphase II (mII) oocytes from which each spindle was microsurgically dissected. Immediately following fertilization, cytokinesis produces a 1-cell embryo (zygote) and associated spindle-enriched second polar body (Pb2) whose transcript profiles also differed one from the other, partially reflecting the enrichment of spindle-associated transcripts. Non-uniform transcript distribution within zygotes did not lead to programmed transcriptome asymmetry between the blastomeres of nascent two-cell embryos, or between the second mitotic products of 3-cell embryos. These findings suggest that mammalian oocytes and zygotes exhibit transcript regionalization without subsequent transcriptome asymmetries between respective early mitotic products. This contrasts the situation in Xenopus and places constraints on the ability of maternal transcriptomic prepatterning to prescribe early mammalian development. 16 Zygote vs. Polar body pairs, 10 spindle vs. oocyte pairs, 22 2-cell embryos and 8 3-cell embryos were analysed. Although the raw data are two channel, only the Cy5 signal of each file was analyzed. The Cy5 channel for each gene is normalized to the average Cy5 intensity of the gene across all samples.

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First cleavage is a manifestation of the geometry of the unfertilized oocyte: implications for monozygotic twinning in mice.

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