Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Knockdown of Zona Pellucida protein 2 (ZP2) in mouse embryos by Trim-away and morpholino combined

ABSTRACT: The zona pellucida (Zp) genes are quintessential oocyte-specific genes that are transcribed only during oogenesis and whose protein products are secreted in the extracellular space. In mice the Zp genes are three: Zp1, Zp2, Zp3 (Zp4 is a pseudogene). Contrary to longtime knowledge, data from 2023 have begun to challenge the notion that the functions of the Zp proteins operate solely in the extracellular space. Doubts began to arise after the results of proteasomal depletion of ZP3 with the Trim-away method (PMID 29153837): mouse zygotes microinjected with a specific antibody anti-ZP3 together with the ubiquitin-protein ligase TRIM21 died within a few hours (PMID 37930049). Given this background, we designed the present study to test whether the knockdown of another protein of the zona pellucida, namely ZP2, would impinge on preimplantation development and transcriptome composition, as previously observed for ZP3. Unlike our previous analysis (GSE278859) this time we have taken into account the additional information that the mRNAs of Zp genes are associated with the ribosomes of pre-implantation mouse embryos (PMID 35697785), which suggests that ZP proteins may be produced de novo after oogenesis. If that were the case, then the depletion of ZP2 by Trim-away alone would not suffice, as the protein amount depleted could be regenerated by new protein synthesis of ZP2. Therefore, this time we combined Trim-away with antisense oligo-mediated inhibition of Zp2 mRNA translation (morpholino). Pronuclear-stage mouse oocytes were microinjected with a cocktail of Trim21-mRNA, antibody anti-ZP2, and morpholino anti-ZP2, or with a cocktail in which antibody and morpholino were both directed against a protein that is not present in wildtype embryos (green fluorescent protein, GFP). We examined three experimental groups the blastocyst stage, in triplicate (R1, R2, R3), as follows. GROUP 1: blastocysts from pronuclear-stage oocytes that were microinjected a cocktail composed of Trim21 mRNA, fluorescent dextran beads, antibody anti-ZP2 and morpholino anti-Zp2, forming a group named “ZP2 knockdown\" (ZP2 KD R1, R2, R3). GROUP 2: blastocysts from pronuclear-stage oocytes were microinjected with a cocktail composed of Trim21 mRNA and fluorescent dextran beads, and cultured further, forming a group named “micromanipulation control\" (MC R1, R2, R3). GROUP 3: blastocysts from pronuclear-stage oocytes were microinjected with a cocktail composed of Trim21 mRNA, fluorescent dextran beads, antibody anti-GFP and morpholino anti-GFP, and cultured further, forming a group named “mock knockdown" (mock KD R1, R2, R3). Approximately eighty hours after microinjection, at the early blastocyst stage, embryos were collected and lysed for transcriptome analysis. GROUPS 1 and 3 were compared with GROUP 2 for mRNAs that are differently expressed (p<0.05; t test); then, the two sets of differently expressed genes were intersected by Venn diagram to identify the genes that are uniquely altered by knockdown of ZP2. These genes that were uniquely altered after ZP2 depletion were subjected to overrepresentation analysis using Webgestalt. Collectively, these data support a conclusion that ZP2 found inside the embryo was not merely a passive remnant from oogenesis, but also served an active functional role during mouse preimplantation development.

INSTRUMENT(S): Illumina NovaSeq X

ORGANISM(S): Mus musculus

SUBMITTER: Michele Boiani

PROVIDER: E-MTAB-16339 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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 + 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.

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: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).

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Knockdown of Zona Pellucida protein 2 (ZP2) in mouse embryos by Trim-away and morpholino combined

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