P-257 Nanosphere-based assessment of zona pellucida permeability in humans and mammalian models
Authors: Guerrero-Sánchez J , Lamas-Toranzo I , González-Brusi L , Pérez-Gómez A , Querejeta-Fernández A , Cabello Y , José H , Bermejo-Alvarez P 
Published: June 2022

Abstract

Study question
What is the maximum particle size that can traverse through human zona pellucida (ZP) and that of other mammals?

Summary answer
Human oocytes ZP is impermeable to 40-100 nm nanospheres. ZP of other mammals is impermeable to 100 nm particles, and permeability changes along preimplantation development.

What is known already
ZP is a porous structure that protects oocytes and early embryos, being permeable to nourishing and signaling substances while blocking the entrance of immune cells and viruses into the perivitelline space. Size-dependent ZP permeability is poorly known and may differ between species, as in humans and rabbits ZP it is composed by 4 proteins (ZP1-4), while bovine and murine ZPs contain only 3 proteins (ZP2-4 in bovine, ZP1-3 in mice). Understanding permeability is crucial to determine embryo susceptibility to viral infections or large biological particles such as exosomes, and to assess possible damages inferred by artificial reproductive techniques.

Study design, size, duration
The objective has been to determine ZP permeability in mice, bovine, rabbit (wild-type or lacking ZP4) or human oocytes and/or embryos based on particle size. The assay was performed in 3 replicates of 3 samples per stage: mouse samples (oocytes, zygotes, 2-cell embryos and blastocysts) collected in vivo, bovine samples (oocytes, zygotes, 4-8 cell embryos and blastocysts) produced in vitro, rabbit samples (zygotes) collected in vivo and immature human oocytes discarded for clinical use.

Participants/materials, setting, methods
Permeability test consisted of incubating oocytes and embryos in 0.005 % solutions of carboxylate-modified polystyrene nanospheres (20, 40 and 100 nm) loaded with a fluorophore emitting at a 505/515 nm for 30 min. Following incubation and washing, samples were observed under a structured illumination fluorescence microscope to determine if particles are present inside ZP (i.e., ZP is permeable to that size of particle) or fluorescence cannot be detected (i.e., particles unable to traverse ZP).

Main results and the role of chance
ZPs of all species and stages blocked efficiently the pass of 100 nm-sized nanospheres. 40 mm nanospheres were blocked by the ZP of oocytes and zygotes in all species with the exception of rabbit zygotes lacking ZP4. ZP permeability changed during early development, as ZP from murine and bovine cleaved embryos and blastocyst were permeable to 40 nm-sized particles. Finally, with the exception or WT rabbit zygotes, ZP from all oocytes and embryos analyzed were permeable to 20 nm nanospheres. As a fluorescence control, murine and bovine oocytes without ZP were exposed to nanospheres of all sizes, which were clearly visualized attached to the oolema or into the ooplasm.

Limitations, reasons for caution
Experiments on cleavage and blastocysts stages were performed on animal models, so whether human ZP permeability changes following fertilization remains unknown.

Wider implications of the findings
Mammalian ZP acts as a selective barrier during early embryo development. Understanding particle size-dependent permeability is crucial to determine embryo susceptibility to viral infection or to signaling mediated by large particles such as extracellular vesicles and exosomes and to assess the impact of oocyte manipulation techniques.

Trial registration number
Not applicable