O-238 Critical requirement of intracellular zona pellucida proteins for mouse embryo survival
Authors: Boiani M , Drexler H , Israel S 
Published: June 2022

Abstract

Study question
Are the intracellular deposits of zona pellucida (ZP) proteins relevant to embryonic development?

Summary answer
Contrary to longstanding views, ZPs are not only oocyte-specific proteins with extracellular tasks, but also have intracellular tasks that are essential for embryo survival.

What is known already
Mutation studies always defined the ZP functions as extracellular, namely: to encase oocytes in ovarian follicles, to ensure species-specific sperm binding, and to dampen shear stress on the embryo surface. Therefore, ZP mutations cause primary infertility due to empty follicles, polyspermic fertilization or harmful contact between embryo and oviductal epithelium. However, when these limitations were obviated in mice by monospermic fertilization in vitro and blastocyst transfer to uterus, the concepti of ZP2-null and ZP3-null oocytes were still unviable (PMID 11245577). This suggests that the tasks of ZPs don’t end in the extracellular space as previously assumed.

Study design, size, duration
After monospermic fertilization, wild-type mouse oocytes were depleted of intracellular ZP proteins using the ‘Trim-away’ method (PMID 29153837). ZP1-3 were targeted by prevalidated antibodies (producing a dominant band in Western blot) and committed for proteasomal degradation by the ubiquitine ligase Trim21. Antibodies were either folded or denatured (heat-inactivated), thereby allowing for efficient or abolished depletion, respectively (negative control). The two groups were examined for development and efficiency of ‘Trim-away’ at degrading the ZPs.

Participants/materials, setting, methods
Mature B6C3F1 female mice were stimulated with gonadotropins (eCG, hCG) and mated to CD1 stud males to collect fertilized superovulated oocytes. These were microinjected with a cocktail of mCherry-Trim21 mRNA and anti-ZP antibody, followed by culture in KSOM(aa) medium for 96 hours. Successful depletion of ZP proteins was measured by mCherry fluorescence, ZP Western blotting and mass spectrometry. Developmental rates were compared between the groups of the folded vs. denatured antibodies.

Main results and the role of chance
Fertilized oocytes depleted of ZP3 did not cleave and remained arrested at 1-cell stage (0 blastocysts / 300 oocytes; 7 replicates). Oocytes depleted of ZP2 cleaved once or twice, and remained arrested at 2- or 4-cell stage (0 blastocysts/ 100 oocytes; 3 replicates). These failures stood in contrast to the blastocyst progression of oocytes depleted of ZP1 (42 blastocysts / 100 oocytes; 3 replicates) and negative controls injected with either of the denatured antibodies (&gt;50% blastocysts) (p &lt; 0.01; chi test). Molecular efficacy of ‘Trim-away’ was manifest from the decline of mCherry fluorescence, and from the reduction of the ZP signal intensity in Western blot and mass spectrometry; whereas the negative controls remained bright-fluorescent and retained the dominant band in Western blot. Inspection of publicly available datasets from two-hybrid and affinity capture screens reveals that ZP3 has more than 20 interaction partners in cell lines. Collectively, this information supports that ZP proteins are required for developmental competence not only outside but also inside of oocytes, where the ZPs might interact with more proteins than just ZP1, ZP2 and ZP3. Our study provides novel insight into the pathogenic mechanism of ZP gene mutations, adding a novel intracellular route to the longstanding extracellular roles.

Limitations, reasons for caution
Although there is sufficient evidence for expanding on the tasks of ZPs in mice, human oocytes contain ZP4 in addition to ZP1-3. While the novel tasks in mice rely on stable protein deposits that persist from oocyte to blastocyst, it is not known if the human counterparts persist that long.

Wider implications of the findings
ZPs may qualify as ‘moonlighting proteins’ that enrich - but at the same time also complicate - the determination of primary infertility phenotype from genotype.

Trial registration number
not applicable