Journal: Nucleic Acids Research

Article Title: EXOSC10 sculpts the transcriptome during the growth-to-maturation transition in mouse oocytes

doi: 10.1093/nar/gkaa249

Figure Lengend Snippet: Exosc10cKO oocytes exhibit dysregulated transcriptomes during oocyte maturation. (A) Schematic illustrating the pipeline of single oocyte RNA-seq. After individual oocyte lysis, oligo-dT beads captured poly(A) RNAs for library construction and sequencing. Genotypes were determined from genomic DNA. (B) Total RNA levels were normalized for each library by an ERCC RNA spike-in mix. (C) Further normalization of total RNA level in B by the mean value of the GV stage within each genotype. (D) Heatmap of all libraries, each row represents one gene and each column represents one library. Genes are ranked from highest to lowest expression level, and every 100th gene from the top half were selected to represent the transcriptome. The transcription level was color-coded from high to low. (E) PCA of the 64 libraries. Each dot represents one library, color-coded by genotype and stage. (F) Log2 fold change of Exosc10 and Gapdh in cKO versus control oocytes. The bars and lines are log2 fold change and standard error of the mean from DESeq2 analyses. **** P-adjust <0.0001, n.s. no significance, which are the P-adjust values in DESeq2 analysis. (G andH) MA-plots of transcript changes from GV to GV3h, and from GV3h to MII stage in control oocytes. The more and less abundant transcripts in each comparison are labeled by red and blue, respectively (both have P-adjust <0.01). (I) MA plot of transcript change from GV3h to MII stage in control oocytes when analyzed by median ratio normalization. (J–L) MA-plots of transcript changes in cKO versus control oocytes at GV, GV3h and MII stages. The increased and decreased abundant transcripts in each comparison are labeled by red and blue, respectively (P-adjust <0.01). (M andN) Gene ontology of the more abundant transcripts in G, J and K.

Article Snippet: The nude oocytes were collected for further experiments. cRNA in vitro transcription and micro-injection The Exosc10 coding sequence was inserted into plasmid #44118 (Addgene) to form an in-frame fusion with mVenus.

Techniques: RNA Sequencing Assay, Lysis, Sequencing, Expressing, Labeling

Journal: Nucleic Acids Research

Article Title: EXOSC10 sculpts the transcriptome during the growth-to-maturation transition in mouse oocytes

doi: 10.1093/nar/gkaa249

Figure Lengend Snippet: Oocyte-specific knockout of Exosc10 causes female subfertility by impairing GVBD during oocyte maturation. (A) Schematic of strategy to generate an Exosc10 floxed allele using CRISPR/Cas9. Two loxP sites were inserted to bracket exons 4–10. (B) Mating strategy to obtain oocyte-specific conditional knockouts of Exosc10 (cKO). Siblings with other genotypes were used as controls. The paternal allele is labeled in blue and the maternal allele is labeled in magenta in the offspring. Note that the floxed maternal Exosc10 allele will become a deletion allele (−) during oocyte growth. (C) qRT-PCR of Exosc10 in single oocytes obtained from controls and cKO mice. Error bars: standard deviation of three technique replicates of each sample. (D) Dot plot of individual litter sizes over 6 months of harem breeding of controls and cKO females with wild-type males. The sizes of the dots are normalized by the average litter number per female. The number in parenthesis is the number of females having the indicated genotypes. The number of pups born is indicated below each group. The horizontal lines represent the mean and standard deviation. (E) Bright-field images of cKO and control oocytes cultured ex vivo for 0 (GV) or 3 h (GV3h). (F) Percentage of GVBD oocytes in E. Numbers of oocytes are indicated below each group. (G and H) Confocal fluorescence and DAPI images of oocytes after lamin B immunostaining at GV (G) and GV3h (H) stages. Lamin B and DAPI are maximum intensity projections. Quantification of lamin B fluorescence is on the right. The horizontal lines inside the violins represent the median and the quartiles. The number of oocytes from at least three females are indicated below each group. **** P < 0.0001 in D, G, H, two-tailed Student's t-test. Scale bars: 100 μm in E; 20 μm in G and H.

Article Snippet: The nude oocytes were collected for further experiments. cRNA in vitro transcription and micro-injection The Exosc10 coding sequence was inserted into plasmid #44118 (Addgene) to form an in-frame fusion with mVenus.

Techniques: Knock-Out, CRISPR, Labeling, Quantitative RT-PCR, Standard Deviation, Cell Culture, Ex Vivo, Fluorescence, Immunostaining, Two Tailed Test