Trim-away depletion of Zona Pellucida 3 (ZP3) protein in mouse zygotes (analysis after 10h)
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. Overall design: Biological replicates were analyzed for each of two experimental conditions: 'Trim-away of ZP3' (protein degradation) in triplicate (r1, r2, r3) and ‚no Trim' (Trim-away cocktail containing the buffer of the antibody in place of the antibody) in triplicate (r1, r2, r3). Trim-away followed to the coinjection of antibody anti ZP3 (Proteintech 21279-1-AP; 1 mg/mL), mCherry-Trim21 mRNA (0.2 mg/mL) and oregon green dextran beads (0.02 mg/mL), resulting in protein degradation in the proteasome. No Trim-away reaction took place after coinjection of mCherry-Trim21 mRNA (0.2 mg/mL), ZP3 antibody buffer, and oregon green dextran beads (0.02 mg/mL). RNA was extracted and analyzed at 10 hours past injection, when the embryos were at the pronuclear stage.