LAUSR

What does a secreted oocyte-specific protein, ZP2, do inside mouse embryos during cleavage? Contrary to prevalent notion, ZP2 protein synthesis is not silenced after ovulation, but goes on in preimplantation embryos to support the morula-blastocyst transition A tenet of reproductive biology is that the expression of oocyte-specific genes must remain silent in embryos. Among such genes, those of the ZP encode glycoproteins that are secreted until end of oogenesis and tasked with extracellular roles during folliculogenesis and fertilization. No further synthesis of ZP proteins is thought to occur after ovulation. However, already since the mouse gene knockout was reported in 2001 it was noticed that ZP2 null oocytes failed to develop to term even when fertilized correctly (PMID 11245577). Likewise, rabbit oocytes in which ZP4 was mutated at pronuclear stage featured defective embryogenesis (PMID 31635692). This is a treatment vs control study conducted in wild-type mice. For profiling of ZP2 protein expression,zygotes were produced in vivo and cultured 4 days in the presence of natural vs isotopic amino acids, and compared to embryos grown in vivo. For analysis of ZP2 function, zygotic knock-down of ZP2 was compared with that of a protein not present in wild-type embryos (green fluorescent protein, GFP), followed by transfer of both groups to uterus. B6C3F1 female mice were stimulated with gonadotropins and mated to CD1 males to produce zygotes. These were labeled continuously in vitro or in vivo using isotopic Arginine + Lysine, and sampled at consecutive stages for monoclonal immunofluorescence and mass spectrometry. To achieve knockdown of ZP2 during embryogenesis, zygotes were microinjected with Trim-Away reagents (PMID 29153837) and/or morpholino oligonucleotide anti-ZP2, followed by scoring of cleavage rates, blastocyst quality (germ layers) and transcriptome and proteome compositions. The most abundant protein of the mouse zona pellucida, ZP2, not only was still present inside embryos up to blastocyst, but also became significantly more abundant in the 4-8 cell stage, as determined by direct immunofluorescence with monoclonal antibody (+25%, p < 0.05, Wilcoxon test). Mass spectrometry after isotopic labeling of protein synthesis during embryo culture confirmed that ZP2 incorporated the label, meaning: de novo protein synthesis of an oocyte-specific gene product in embryos. Zygotic knockdown via morpholino oligonucleotides and Trim-Away resulted in 50% reduction of ZP2 abundance at the 8-cell stage (p < 0.05, Wilcoxon test), accompanied by growth retardation at the 8-cell and morula stage. Embryos with depletion of ZP2 formed blastocysts with significantly smaller trophectoderm and primitive endoderm compartments. These effects were not observed in controls subjected to mock knockdown (GFP). Consistent with these morphometric data, transcriptome and proteome analyses returned ‘embryonic lethality’ and ‘embryonic growth retardation’ among the over-represented gene ontology terms of ZP2 (but not GFP) knockdown embryos. Thus, intra-embryonic ZP2 appears necessary to keep up the pace of cleavage progression and secure blastocyst fitness. This discovery can explain the historical conundrum of why ZP2 null oocytes formed blastocysts and yet these could not develop to birth (PMID 11245577). Although there is sufficient evidence for expanding on the tasks of ZP proteins in mouse development, human oocytes contain ZP4 in addition to ZP1-3. While the novel tasks of ZP2 in mice hinge on de novo protein synthesis, it is unclear if this occurs also in humans. Our study provides novel insight into the pathogenic mechanism of ZP gene mutations, adding a novel intracellular route to the longstanding extracellular roles. If ZP proteins function inside human oocytes and embryos, then using ICSI to overcome problems of thick or thin zona pellucida remains ineffective. No