{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"submitter":["Michele Boiani"],"organism":["Mus musculus"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-16339"],"description":["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."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Sequencing - Double-read sequencing with a read length of 111bp was performed on NovaSeq® X Plus System  using the corresponding NovaSeq® X Series 1.5B Reagent Kit (200 Cyc).","Sample Collection - Approximately 80 hours after microinjection of Trim-away cocktail into pronuclear-stage oocytes, embryos were collected from the culture medium (KSOM with amino acids) at the blastocyst stage. Nine (9) blastocysts, zona-intact, were allocated to each sample.","Nucleic Acid Extraction - RNA was extracted and purified using Quick-RNA MicroPrep (cat. no. R1051, Zymo Research Europe GmbH).","Library Construction - The library preparation of the total RNA was performed with the Watchmaker mRNA Library Prep Kit (7K0078-024) according to the manufacturer’s instructions. Total RNA integrity and quality of the library were assessed using a TapeStation4200 (Agilent, Santa Clara, CA, USA).","Growth Protocol - Zygotes were cultured in KSOM medium containing amino acids in 4-well plates, at 37 °C under 6 % CO2 in air.","Sample Treatment - Pronuclear-stage oocytes were microinjected with Trim-away + morpholino cocktail (directed against either ZP2 or GFP) as treatment, or with sole Trim21 mRNA as control."],"figure_sub":["Organization","MINSEQE Score","Assays and Data","Processed Data","MAGE-TAB Files"],"data_protocol":["Data Transformation - Counts were used to calculate the FPKM to normalize for sequencing depth and gene length. FPKM values for Ensembl gene identifiers corresponding to the same gene symbol were averaged.","Sequence Alignment - Reads were aligned to the reference genome (Mus musculus Ensembl GRCm39) using Hisat2 (PMID 25751142; version: 2.2.1). Aligned reads were sorted using samtools (PMID 21903627; version 1.16.1). Sorted and aligned reads were counted into genes using HTSeq framework (PMID 25260700; version 2.0.3), then these counts were used to calculate the FPKM to normalize for sequencing depth and gene length. StringTie version 3.0.0. was used to calculate the potential transcripts."],"omics_type":["Metabolomics","Unknown","Transcriptomics","Genomics","Proteomics"],"instrument_platform":["Illumina NovaSeq X"],"pubmed_abstract":["The zona pellucida (ZP) is the quintessential extracellular structure of mammalian oocytes. Contrary to long-standing view that the synthesis of ZP proteins is specific to oocytes and muted in embryos, we report here that the major zona pellucida protein ZP2 is re-synthesized and functionally required during mouse embryo development. The orthogonal methods of mass spectrometry and monoclonal immunofluorescence revealed an increase of ZP2 abundance at the 8-cell / morula stage, which did not occur when zygotes were microinjected with translation-blocking oligonucleotides (morpholinos). To shed light on the functional significance of embryonic ZP2, we performed protein knockdown using immunodepletion (by ‘Trim-Away’) while at the same time preventing replenishment (by translation-blocking morpholino). ZP2 knockdown resulted in morula stage retardation and formation of defective blastocysts, whose cell lineages trophectoderm and primitive endoderm were smaller and less able to support post-implantation development. The transcriptional correlates of these morphological alterations had a gene ontology (biological process) signature that included cell lineage-relevant terms (‘endoderm development’, ‘gastrulation’), while the proteomic correlates had a gene ontology signature related to protein synthesis. Taken together, these results call into question the traditional model that ZP proteins function solely in the extracellular space and accompany embryogenesis as passive bystanders: on the contrary, ZP proteins also participate actively in the intracellular processes of early embryogenesis."],"study_type":["RNA-seq of coding RNA"],"species":["Mus musculus"],"pubmed_title":["A novel and critical role of the intracellular Zona Pellucida protein 2 (ZP2) for blastocyst formation in mice"],"pubmed_authors":["Michele Boiani","Thomas Nolte, Steffen Israel, Hannes C.A. Drexler, Georg Fuellen, Michele Boiani"],"additional_accession":[]},"is_claimable":false,"name":"Knockdown of Zona Pellucida protein 2 (ZP2) in mouse embryos by Trim-away and morpholino combined","description":"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.","dates":{"release":"2025-12-19T00:00:00Z","modification":"2026-05-30T15:55:23.263Z","creation":"2025-12-01T17:45:34.359Z"},"accession":"E-MTAB-16339","cross_references":{"ENA":["ERP185912"],"EFO":["EFO_0002944","EFO_0004170","EFO_0003789","EFO_0004917","EFO_0005518","EFO_0003816","EFO_0003738","EFO_0004184","EFO_0003969"],"doi":["10.64898/2025.12.12.692802"]}}