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