Project description:Study of the interaction between Me31B and UBC-E2H (Kdo) in stage 14 Drosophila oocytes as part of a study of RNA-binding proteins during the maternal-to-zygotic transition
Project description:Mature stage 14 oocytes prior to egg activation were isolated from wild type Oregon Red fly cultures by a combined blender/ sieving method which allows specific and efficient enrichment of stage 14 oocytes. Total RNA was extracted from 4 replicate collections and submitted to Affymetrix microarray hybridisation.
Project description:Global regulation of spindle-associated proteins is crucial in oocytes due to the absence of centrosomes and their very large cytoplasmic volume, but little is known about how this is achieved beyond involvement of the Ran-importin pathway. We previously uncovered a novel regulatory mechanism in Drosophila oocytes, in which the phospho-docking protein 14-3-3 suppresses microtubule binding of Kinesin-14/Ncd away from chromosomes. Here we report systematic identification of microtubule-associated proteins regulated by 14-3-3 from Drosophila oocytes. Proteins from ovary extract were co-sedimented with microtubules in the presence or absence of a 14-3-3 inhibitor. Through quantitative mass-spectrometry, we identified proteins or complexes whose ability to binding microtubules is suppressed by 14-3-3, including the chromosomal passenger complex (CPC), the centralspindlin complex and Kinesin-14/Ncd. We showed that 14-3-3 binds to the disordered region of Borealin, and this binding is regulated differentially by two phosphorylations on Borealin. Mutations at these two phospho-sites compromised normal Borealin localisation and centromere bi-orientation in oocytes, showing that phospho-regulation of 14-3-3 binding is important for Borealin localisation and function.
Project description:We aimed to quantify in silico the expression level of candidate genes in different Clytia gonad tissues and oocytes at different growth stage
Project description:We aimed to reveal gene expression profiles of human oocytes at each maturation stage by single cell RNA-seq analyses. We investigated transcriptomes of immature oocytes at the germinal vesicle (GV) stage, maturating oocytes at the metaphase I (MI) stage and in vitro matured oocytes at the metaphase II (MII) stage.
Project description:We provide complete translatome of mouse oocytes from aged females after the breakdown of nuclear envelope (NEBD). RNAseq of polysomal and non-polysomal RNA was determined by using a novel approach combining polysomal fractionation of 200 NEBD-staged oocytes in small SW55Ti centrifugation tubes in the absence of any external spike and qRT-PCR analysis of ribosomal RNA in collected fractions. We compared these data to the translatome of same stage oocytes from young females (GSE121358). We report that genome-wide translatome profiling reveals considerable numbers of transcripts that are differentially translated in oocytes obtained from aged compared to young females. Additionally, we show the majority of aberrantly translated mRNAs in older oocytes have roles associated with cell cycle regulation. Indeed, we demonstrate the differential translation rates of four specific maternal age related transcripts (Sgk1, Castor1, Aire and Eg5) are associated with distinct localization patterns around the newly forming meiotic spindle.
Project description:The meiotic spindle in oocytes is formed without centrosomes. How a bipolar spindle is assembled and maintained around chromosomes in oocytes remains to be established. Multiple kinases are known to regulate the meiotic spindle, but how they execute their function is poorly understood. We found that the phospho-docking protein 14-3-3ε, together with the other isoform ζ, stabilises spindle bipolarity in Drosophila oocytes. A critical 14-3-3 target is the minus-end directed motor Ncd (human HSET; kinesin-14) which has well documented roles in stabilising a bipolar spindle in oocytes. Phospho-docking by 14-3-3 inhibits the microtubule binding activity of the non-motor Ncd tail. Further phosphorylation by Aurora B kinase can release Ncd from this inhibitory effect of 14-3-3. As Aurora B localises to chromosomes and spindles, 14-3-3 facilitates specific association of Ncd with spindle microtubules by preventing Ncd from binding to non-spindle microtubules in oocytes. Therefore, 14-3-3 translates a spatial cue provided by Aurora B to target Ncd selectively to the spindle within the large volume of oocytes.
Project description:To explore the impact of BTG4-T145/S146/S147 mutations on the degradation of transcripts in MII-stage oocytes, we injected wild-type BTG4 mRNA (BTG4wt) and mutant BTG4 mRNA (BTG4tm) into GV-stage oocytes. These oocytes were matured in vitro to the MII stage, after which samples of MII-stage oocytes were collected for library construction and subsequent second-generation transcriptome sequencing.
Project description:Mouse oocytes and embryos from the germinal vesicle (GV) stage to the blastocyst stage were collected and treated with acidic Tyrode’s solution to remove the zona pellucida. We used 42.5 oocytes or embryos per sample at each developmental stage and identified the proteome during mouse pre-implantation development by label-free quantification.
Project description:We performed mouse single oocyte RNA-seq and bulk oocyte CUT&Tag assays in the current project. In details, SMART-seq based single oocyte RNA-seq was performed at adult GV stage, GV3h stage and MII stage, using control and Dis3 oocyte-speicfic knockout (cKO) oocytes. RiboMinus-seq based single oocyte RNA-seq was performed at adult GV stage using control and Dis3 cKO oocytes, and at p20 GV stage using control, Dis3 cKO, Exosc10 cKO and Dis3/Exosc10 double cKO (dcKO) oocytes. Bulk CUT&Tag of anti-H3K27me3 was done in WT and Dis3 cKO oocytes at adult GV stage, and in WT and Dis3/Exosc10 dcKO oocytes at p20 stage. In addition, CUT&Tag of anti-RNA polymerase II (Ser2+Ser5) was performed in WT and Dis3 cKO oocytes at GV stage. All CUT&Tag experiments share the same rabbit-Igg negative control. All p20 stage oocytes were specified as p20. The non-specified GV oocytes were all adult GV oocytes.