Journal: Molecular Human Reproduction

Article Title: Intracellular fraction of zona pellucida protein 3 is required for the oocyte-to-embryo transition in mice

doi: 10.1093/molehr/gaad038

Figure Lengend Snippet: Fertilized mouse oocytes (zygotes) used in the epitope masking and knockdown (Trim-away) experiments.

Article Snippet: In the early dataset, samples comprised 13 embryos in each of two groups: group 1 injected with mCherry-Trim21 mRNA, OGDB, and buffer of the ZP3 Proteintech antibody (triplicate), and group 2 injected with mCherry-Trim21 mRNA, OGDB, and anti-ZP3 Proteintech antibody (triplicate).

Techniques: Knockdown, Microinjection

Journal: Molecular human reproduction

Article Title: Intracellular fraction of zona pellucida protein 3 is required for the oocyte-to-embryo transition in mice.

doi: 10.1093/molehr/gaad038

Figure Lengend Snippet: Figure 6. Degradation of ZP3 in mouse zygotes via Trim-away exacerbates the effect of epitope masking by antibody alone. (a) Representative images of zygotes that not only fail to cleave but also degenerate when anti-ZP3 antibody (Proteintech 21279-1-AP) is coinjected with mCherry-Trim21 mRNA (Trim-away). mCHERRY is the fluorescent tag of TRIM21. (b) Developmental rates of the zygotes subjected to Trim-away or its controls, e.g. microinjection of mCherry-Trim21 mRNA only or mRNA with antibody buffer (third flow-through of the Amicon filter device). In each of the five series, the multiple bars next to each other stand for the replicates (mCherry-Trim21 mRNA only, six replicates; ZP3 Trim-away, eight replicates; antibody bufferþmCherry-Trim21 mRNA, five replicates; ZP1 Trim-away, two replicates; ZP2 Trim-away, three replicates) and the rates of development were normalized to the one-cell stage (100%). For the replicates of the same group, the color is the same, but in different shades. Trim-away of ZP3 hampered the first cleavage, Trim-away of ZP2 hampered the second cleavage, while Trim-away of ZP1 allowed for blastocyst formation but the blastocysts were consistently smaller and less robust than controls. The complete dataset and its breakdown into replicates are provided in Table 1. Square brackets between the two series of ZP3 Trim-away and antibody buffer indicate the statistical comparison and its P-value (Wilcoxon test). ZP, zona pellucida; TRIM21, tripartite motif containing 21.

Article Snippet: Degradation of ZP3 in mouse zygotes via Trim-away exacerbates the effect of epitope masking by antibody alone. (a) Representative images of zygotes that not only fail to cleave but also degenerate when anti-ZP3 antibody (Proteintech 21279-1-AP) is coinjected with mCherry-Trim21 mRNA (Trim-away). mCHERRY is the fluorescent tag of TRIM21. (b) Developmental rates of the zygotes subjected to Trim-away or its controls, e.g. microinjection of mCherry-Trim21 mRNA only or mRNA with antibody buffer (third flow-through of the Amicon filter device).

Techniques: Microinjection, Comparison

Journal: Molecular human reproduction

Article Title: Intracellular fraction of zona pellucida protein 3 is required for the oocyte-to-embryo transition in mice.

doi: 10.1093/molehr/gaad038

Figure Lengend Snippet: Figure 7. Effects of ZP3 knockdown on the mouse embryonic transcriptome. (a) Principal component analysis (PCA) of the zygotes that were subjected to transcriptome analysis 10 h after microinjection of mCherry-Trim21 mRNA with anti-ZP3 (Proteintech 21279-1-AP) or its buffer. (b) At 24 h, a new dataset was generated (main) in which zygotes were subjected to transcriptome analysis same as in (a) except that the time between microinjection and sampling was longer. A third group of non-injected zygotes was also included. For these three conditions, the Venn diagram describes the overlap in detected transcripts at 24 h. The RNAseq datasets are available in Gene Expression Omnibus (GSE232142, 10h; GSE203626, main) and the main dataset is also provided in simplified form in Supplementary Table S2. (c) PCA based on the core of 11 137 transcripts that are common to all samples of the main dataset. Each point corresponds to one sample (mCherry-Trim21 mRNA with antibody buffer in triplicate, ZP3 Trim-away in quadruplicate, non-manipulated in unicate). The first two principal components (PCs) of the data are represented. The PCA of the transcriptome resolves the ZP3 Trim-away and the other groups in the second component. (d) Volcano plot showing the effect of ZP3 Trim-away on the embryo transcriptome at the chronological two-cell stage compared to mCherry-Trim21 mRNA plus antibody buffer (24 h dataset). The numbers of mRNAs that are under- versus over-expressed (fold change >2, adj.P-value < 0.05, t-test) are shown in the upper corners. (e) The 198 differently expressed mRNAs were subjected to GO analysis using Enrichr (Chen et al., 2013), and the top-10 terms (ranked by P-value) were visualized for three ontologies: GO ‘biological process’, GO ‘cellular component’, and mammalian phenotype ontology. ZP, zona pellucida; GO, gene ontology.

Article Snippet: Degradation of ZP3 in mouse zygotes via Trim-away exacerbates the effect of epitope masking by antibody alone. (a) Representative images of zygotes that not only fail to cleave but also degenerate when anti-ZP3 antibody (Proteintech 21279-1-AP) is coinjected with mCherry-Trim21 mRNA (Trim-away). mCHERRY is the fluorescent tag of TRIM21. (b) Developmental rates of the zygotes subjected to Trim-away or its controls, e.g. microinjection of mCherry-Trim21 mRNA only or mRNA with antibody buffer (third flow-through of the Amicon filter device).

Techniques: Knockdown, Microinjection, Generated, Sampling, Injection, Gene Expression

Journal: Molecular human reproduction

Article Title: Intracellular fraction of zona pellucida protein 3 is required for the oocyte-to-embryo transition in mice.

doi: 10.1093/molehr/gaad038

Figure Lengend Snippet: Figure 8. Effects of ZP3 knockdown on cytoplasmic translation and distinct effects of the knockdown depending on the early versus late phase of the two-cell stage in mice. (a) Zygotes were preloaded with mCherry-Trim21 mRNA, and after one cleavage they were microinjected with OGDB or anti- ZP3 (Proteintech 21279-1-AP) in one blastomere only, to then be subjected to analysis of protein synthesis using Click-iTTM OPP Alexa FluorTM 647 imaging kit. Note the reduction of mCHERRY fluorescence in the blastomere receiving anti-ZP3 (white arrows) while the blastomere receiving OGDB retained its fluorescence. OPP fluorescence intensities were measured in both blastomeres of each embryo, and used to calculate absolute interblastomere differences of OPP signal intensity in the Trim-away versus control (OGDB microinjection) groups (P¼0.017; Wilcoxon test). The interblastomere difference is larger in Trim-away than in control, because ZP3 knockdown reduces the protein synthesis in one blastomere. The raw measurement data are provided in Supplementary Table S3. (b) Using the same design as in (a) but delaying the anti-ZP3 injection, the knockdown of ZP3 did not result in blastomere arrest when conducted at the late two-cell stage compared to the early two-cell stage. After 72 h of culture, the blastomere that underwent Trim-away at the early two-cell stage produced no cell progeny (arrows and dotted circle), whereas both blastomeres participated in blastocyst formation when Trim-away was applied at the late two-cell stage. AU, arbitrary units; EGA, embryonic genome activation; OPP, O-propargyl-puromycin, ZP, zona pellucida; OGDB, Oregon Green dextran beads.

Article Snippet: Degradation of ZP3 in mouse zygotes via Trim-away exacerbates the effect of epitope masking by antibody alone. (a) Representative images of zygotes that not only fail to cleave but also degenerate when anti-ZP3 antibody (Proteintech 21279-1-AP) is coinjected with mCherry-Trim21 mRNA (Trim-away). mCHERRY is the fluorescent tag of TRIM21. (b) Developmental rates of the zygotes subjected to Trim-away or its controls, e.g. microinjection of mCherry-Trim21 mRNA only or mRNA with antibody buffer (third flow-through of the Amicon filter device).

Techniques: Knockdown, Imaging, Fluorescence, Control, Microinjection, Injection, Produced, Activation Assay