Project description:In order to study early developmental events in the mammalian embryo it is often desirable to be able to impair expression of specific genes. While DNA and RNA methods are routine, protein methods are still at the very beginning. When a specific antibody is supplied to mouse oocytes expressing the ubiquitin-protein ligase TRIM21, a ternary complex forms with the target protein, leading to its rapid and acute degradation - hence the name 'Trim-away' (PMID 29153837). However, there are many unknowns in this new endeavour. First and foremost, the extent to which endogenous proteins can be depleted depends on their amount, in relation to the amount of exogenous antibody, which is limited by the microinjection procedure. Secondly, the depletion of the protein must be sustained over days. Using the iBAQ algorithm we show that proteins found in preimplantation mouse embryos range from 3,5E-10 to 2,6E-02 picomoles. These amounts are tractable with our microinjection method, which supplies up to 100 picoliters and up to 6,7E-4 picomoles antibody before incurring in toxic effects on mouse development. Building on these data, we demonstrate the feasibility of protein knock-down for a gene which is essential in the preimplantation mouse embryo, namely TEAD4 (TEA domain family member 4). Protein knock-down persists for sufficient time to result in a phenotype which is entirely consistent with that of the null mutation (Tead4 -/-) and of the RNA interference, namely: significantly reduced mRNA expression of TEAD4 target genes Cdx2 and Gata3, and embryo’s inability to implant. We conclude that for a time window of 3-4 days of preimplantation development the protein depletion method can be a valid alternative to DNA and RNA methods. After in vivo fertilization and short culture in KSOM(aa) medium, pronuclear-stage oocytes (B6C3F1 x CD1) were microinjected with Trim21 mRNA and dextran beads as tracer (named 'group 4'), or Trim21 mRNA, dextran beads and anti-GFP antibody (named 'group 5'), or Trim21 mRNA, dextran beads and anti-TEAD4 antibody (named 'group 6'), two replicates each ('a' and 'b'). On day 4 after microinjection, the most advanced embryos were examined for phenotype or lysed for transcriptome analysis. TEAD4-depleted embryos formed only 30% blastocysts and these were not able to implant in the outgrowth assay, in contrast to the almost full rates of the embryos injected with Trim21 mRNA or Trim21 mRNA + anti-GFP antibody. Transcriptome analysis revealed that the TEAD4 target genes Cdx2 and Gata3 are significantly reduced in the embryos that received the anti-TEAD4 antibody compared to the embryos that received Trim21 mRNA only, while the embryos that received anti-GFP antibody were much similar to those that received Trim21 mRNA only.

Project description:Mutation studies always defined the functions of the zona pellucida (ZP) as extracellular, namely: to encase the oocytes in ovarian follicles, to ensure species-specific sperm binding, and to dampen shear stress on the embryo surface. Therefore, mutations in the three ZP mouse genes ZP1, ZP2 or ZP3 cause primary infertility due to empty follicles, polyspermic fertilization or harmful contact between embryos and oviductal epithelium. However, the concepti of ZP2-null and ZP3-null oocytes were still unviable also when the defects were obviated by monospermic fertilization in vitro and blastocyst transfer to uterus (PMID 11245577). This suggests that the tasks of ZPs don’t end in the extracellular space as previously assumed, but there may be also intracellular functions yet to be discovered. The present study tested if experimentally induced degradation of intracellular ZP3 impacted on the development and transcriptome of mouse embryos. To this end we degraded ZP3 using its antibody in conjunction with the ubiquitin-protein ligase TRIM21. This method is known as 'Trim-away' (PMID 29153837). Briefly, in this method a cell (e.g. oocyte) expressing TRIM21 is supplied e.g. injected with a specific antibody to a protein of interest, in this case ZP3. As a result, the ternary complex (target protein-antibody-TRIM21) is destroyed in the proteasome. TRIM21 is here always to be understood as translation product of microinjected mCherry-Trim21 mRNA. We compared two experimental groups, as follows. Pronuclear-stage oocytes (B6C3F1 x CD1) were microinjected with approx. 100 picoliters of mix comprised of mCherry-Trim21 mRNA 0.2 mg/mL + anti-ZP3 antibody (Proteintech 21279-1-AP) 1 mg/mL + dextran beads 0.02 mg/mL, forming a group named 'Trim-away ZP3' group, in triplicate. As a reference, pronuclear-stage oocytes were microinjected with the same mixture as above, except that the antibody buffer was used in lieu of the antibody itself, in triplicate, forming a group named ‚no Trim'. To identify differently expressed genes we compared group 'Trim-away ZP3' with group ‘no Trim’. Ten hours after microinjection, embryos were collected and lysed for transcriptome analysis. Transcriptome analysis revealed that embryos of group 'Trim-away ZP3' and group ‘no Trim' differed in gene expression and were resolved in principal component analysis. The data support a conclusion that ZP3 found inside the embryo was not merely a remnant from oogenesis, but served an intracellular, post-fertilization role during mouse preimplantation development.

Project description:Mutation studies always defined the functions of the zona pellucida (ZP) as extracellular, namely: to encase the oocytes in ovarian follicles, to ensure species-specific sperm binding, and to dampen shear stress on the embryo surface. Therefore, mutations in the three ZP mouse genes ZP1, ZP2 or ZP3 cause primary infertility due to empty follicles, polyspermic fertilization or harmful contact between embryos and oviductal epithelium. However, the concepti of ZP2-null and ZP3-null oocytes were still unviable also when the defects were obviated by monospermic fertilization in vitro and blastocyst transfer to uterus (PMID 11245577). This suggests that the tasks of ZPs don’t end in the extracellular space as previously assumed, but there may be also intracellular functions yet to be discovered. The present study tested if experimentally induced degradation of intracellular ZP3 impacted on the development and transcriptome of mouse embryos. To this end we degraded ZP3 using its antibody in conjunction with the ubiquitin-protein ligase TRIM21. This method is known as 'Trim-away' (PMID 29153837). Briefly, in this method a cell (e.g. oocyte) expressing TRIM21 is supplied e.g. injected with a specific antibody to a protein of interest, in this case ZP3. As a result, the ternary complex (target protein-antibody-TRIM21) is destroyed in the proteasome. TRIM21 is here always to be understood as translation product of microinjected mCherry-Trim21 mRNA. We compared two experimental groups, as follows. Pronuclear-stage oocytes (B6C3F1 x CD1) were microinjected with approx. 100 picoliters of mix comprised of mCherry-Trim21 mRNA 0.2 mg/mL + buffer of ZP3 antibody + dextran beads 0.02 mg/mL as tracer, forming a group named 'Trim21 overexpression', in quadruplicate. As a reference, pronuclear-stage oocytes were microinjected with mCherry-Trim21 mRNA 0.2 mg/mL + anti-ZP3 antibody (Proteintech 21279-1-AP) 1 mg/mL + dextran beads, forming a group named 'Trim-away ZP3' group, in triplicate. To identify differently expressed genes we compared group 'Trim-away ZP3' with group 'Trim21 overexpression'. In addition, a single 'non-manipulated' sample was also incuded merely to confirm that Trim21 was detectable in the two experimental groups, but not in the non-microinjected embryos. On the day after microinjection, embryos were collected and lysed for transcriptome analysis. Those of group 'Trim21 overexpression' were at the 2-cell stage so as the non-manipulated embryos, whereas those of group 'Trim-away ZP3' were arrested at the 1-cell stage. Transcriptome analysis revealed that embryos of group 'Trim-away ZP3' and 'group 'Trim21 overexpression' differed in the expression of 197 of 11137 genes (t test, FDR<0.05). The data support a conclusion that ZP3 found inside the embryo was not merely a remnant from oogenesis, but served an intracellular, post-fertilization role during mouse preimplantation development.

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Analysis of TEAD4 protein-depleted mouse embryos after Trim-away

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