Understanding fertility issues in animals

When an egg is fertilised by a single sperm cell, its coat (zona pellucida, ZP) will quickly harden, making the entry of a second sperm cell impossible. This process is tightly controlled in all living beings, but all the mechanisms involved are not well understood. Hardening the egg coat as soon as the first sperm cell enters is crucial, as several sperm cells inside an egg will cause polyspermy which will end the development of the egg. The human ZP is comprised of several ZP proteins. Among them, the ZP2 glycoprotein (a protein that can bind sugar molecules) is one of the main proteins involved in the egg coat composition. Interestingly, this protein is cleaved as a result of the fertilisation. Researchers studied how the cleavage of the ZP2 protein affects the structure of the egg coat.

Photographic image of a light micrograph (Nomarski differential interference contrast) of an unfertilised mouse egg incubated in the presence of free-swimming sperm.
Sperm are shown bound to the zona pellucida. Scale bar = ≃14 µm.

hZP2 is a protein composed of an NTR (N terminal region) with three different parts, N1, N2 and N3 and a ZP module. ZP modules from different ZP proteins will interact and form filaments, and ultimately form a loose net around the unfertilised egg. Researchers completed MX experiments at Diamond to understand the changes in the ZP proteins after fertilisation of the egg. They first studied the shape of the ZP2 Nter region in a pre-fertilisation state which showed that this region of the protein forms a V-shape. The authors found that after cleavage, the protein will form dimers, and the cleavage is regulated by the N1 domain of the Nter region. As Nter regions of different ZP proteins are forming dimers, ZP domains are moving closer. All these different interactions have been identified using the technique of macromolecular crystallography (MX)  collecting data from crystals using X-ray diffraction in synchrotrons in Europe.

The mechanisms linked to infertility

ZP proteins are found in all animals and therefore have a very conserved role in organisms. This study highlights the importance of different regions of the ZP proteins in preventing polyspermy during the first steps of the egg development. As many mutations in the human ZP genes are linked with sterility, the new structures resolved in this study will help to understand the different mechanisms linked with infertility. An important point for this study is the gap between the data collection and the analysis of the data. When the data were collected in 2014, the available software and models at that time were not advanced enough to resolve the structure of ZP filaments. The development of improved software and the prediction of 3D models via Alphafold allowed the correct interpretation of the MX data all these years later after the experiments.