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Trim-away depletion of Zona Pellucida 3 (ZP3) protein in mouse zygotes (analysis after 24h)

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE203626 | GEO | 2023/10/12

REPOSITORIES: GEO

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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:In metazoans, maternal factors deposited in the ooplasm during oocyte growth are largely responsible for the control of initial development of the newly formed embryo. The gradual handover from oocytic to embryonic control, known as oocyte-to-embryo or maternal-to-zygotic transition, involves de novo synthesis of new embryonic factors (embryonic genome activation) as well as the timely degradation of preexisting maternal factors deposited in oocytes as transcripts or proteins. Nevertheless, the role of the protein deposits largely remains undeciphered. In order to study these deposits, genetic knockouts and knockdown systems are not suited because they do not react with the protein that has already been deposited. Targeted proteolysis can be accomplished with the 'Trim-away' method (PMID 29153837). Building on our proteomic datasets and our own adaptation of the Trim-away method (PMID 31638890), we demonstrate a large deposit of COP9 signalosome complex subunit 3 (COPS3) protein in mouse oocytes, and its critical role during the oocyte-to-embryo transition. COPS3 protein depletion in zygotes causes 2-cell arrest, at variance with the original report describing early postimplantation lethality (PMID 12972600). We conclude that peri-implantation demise was possibly the second time when the gene function was needed in development, while earlier functions had been hidden by maternal deposits. In the Trim-away method (PMID 29153837) a cell expressing the ubiquitin-protein ligase TRIM21 is supplied e.g. injected with a specific antibody to a protein of interest. As a result, the ternary complex (target protein-antibody-TRIM21) is destroyed in the proteasome. To test for the requirement of COPS3 in early mouse development, we chose to deplete COPS3 using its antibody in conjunction with TRIM21. To distinguish between depletion and inhibition of the target protein, we also tested the antibody alone. In total, we examined four groups, as follows. Pronuclear (PN)-stage oocytes (B6C3F1 x CD1) were microinjected with 100 picoliters of mix comprised of: Trim21 mRNA 0.2 mg/mL + dextran beads as tracer (named 'group 2'); Trim21 mRNA 0.2 mg/mL + anti-COPS3 antibody 0.5 mg/mL + dextran beads as tracer (named 'group 3'); anti-COPS3 antibody 0.5 mg/mL + dextran beads as tracer (named 'group 4'). Antibody was ab79698 from AbCam, column-purified twice to remove preservatives. These groups were compared with non-injected PN oocytes (named 'group 1'). Each group was produced in duplicate (replicate '.0' and replicate '.1'). On the day after microinjection, late 2-cell embryos were collected and lysed for transcriptome analysis. Zygotes of groups 3 and 4 presented stunted progression to the 2-cell stage, in contrast to the almost full rates of the zygotes of groups 1 and 2. Transcriptome analysis revealed that embryos group 2 are very similar to those of group 1 (97 differently expressed mRNAs), whereas embryos of groups 3 and 4 differ from those of group 1 by 3499 and 973 mRNAs, respectively (fold change ≥2, p≤0.05; Student’s t test).

Project description:An important facet of the oocyte to embryo transition in mammals is the rapid elimination of a subset of the maternal products that got accumulated during oogenesis. RNA studies support a view that the transition occurs quite rapidly. Whether this applies also for proteins remains to be determined beyond the very few cases known to date. We report that COPS3, the 3rd subunit of the COP9 signalosome complex, forms a large protein deposit in mouse oocytes (94th percentile of the riBAQ distribution). The one and only knock-out study had previously shown that Cops3 null (-/-) mouse embryos obtained from heterozygous intercrosses arrested after 5.5 dpc and were resorbed by 8.5 dpc mainly due to increased cell death in the epiblast (PMID: 12972600). However, more recent studies from our group ascribed Cops3 gene with a developmental role already at the 2-cell stage. Specifically, the sister blastomeres differ from each other in Cops3 mRNA levels and distribution, preceding the discordance of epiblast formation in derivative twin blastocysts, and implicating Cops3 in the molecular underpinnings of totipotency. This discrepancy between original knock-out result and recent observations can be resolved if we posit that the first requirement for Cops3 gene function was bridged by maternal protein that outlived the locus excision, consistent with the observation that Cops3 -/- blastocysts had the same immunostaining intensity as the +/- or +/+ counterparts in the original knock-out study. Thus, these contradicting observations support a hypothesis that 5.5 dpc may not have been the first time when the gene function was needed in development, but the second time. To test this hypothesis, pronuclear-stage mouse oocytes were subjected to an immunological method that directly targets the protein of interest by way of proteasomal degradation of the COPS3-antibody-TRIM21 complex. Briefly, pronuclear-stage mouse oocytes were microinjected with a mixture of the COPS3-specific purified monoclonal IgG antibody, mCherry-Trim21 mRNA and Oregon Green dextran beads (inert tracer). Injected oocytes were then allowed to develop and collected for lysis 24 hours later, when they reached the 2-cell stage but were not able to progress further. In addition to the COPS3, we targeted two additional proteins, OCT4 and TEAD4. The following seven experimental groups were examined by liquid chromatography-mass spectrometry (LC-MS/MS) using the pipeline described previously by Israel et al.: 1) non-manipulated 2-cell embryos; 2) 2-cell embryos from oocytes injected with Oregon Green dextran beads; 3) 2-cell embryos from oocytes injected with mCherry-Trim21 mRNA and Oregon Green; 4) 2-cell embryos from oocytes injected with mCherry-Trim21 mRNA, Oregon Green and anti-COPS3 (Abcam 79698); 5) 2-cell embryos from oocytes injected with anti-COPS3 (Abcam 79698) and Oregon Green; 6) 2-cell embryos from oocytes injected with mCherry-Trim21 mRNA, Oregon Green and anti-TEAD4 (Abcam 58310); 7) 2-cell embryos from oocytes injected with mCherry-Trim21 mRNA, Oregon Green and anti-OCT4 (Abcam 181557).

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.

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Trim-away depletion of Zona Pellucida 3 (ZP3) protein in mouse zygotes (analysis after 24h)

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