International Business Department           Liu Bojia           March 18, 2023

  Fertilisation, the first step in the birth of life, is an extremely delicate physiological process. After the first sperm fuses with the egg cell membrane and enters the egg cell, the rest of the sperm, even after a long journey, will not be able to knock on the door of the egg cell.

  Although it may sound cruel to the hard-working sperm, this timely "closing of the door" is important for the continuation of life. In most animals, once more than one sperm has fused with an egg cell, the resulting multi-prokaryotic fertilised egg will end up dead.

  The question is, in what way does the egg cell prevent the second sperm from entering?

  In mammals, sperm first pass through an outer oocyte coat called the zona pellucida (ZP), a mesh-like outer structure that serves to protect the oocyte as well as the early embryo, before touching the oocyte membrane.

  Scientists already know that the human zona pellucida is a filamentous outer membrane made up of a polymerisation of four glycoproteins, ZP1-ZP4, of which the ZP2 protein plays a key role in the sperm-oocyte interaction. After the first sperm enters the oocyte, the oocyte releases cortical granules in which the protease ovastacin cleaves the ZP2 protein at specific sites, making the zona pellucida hard and impossible for subsequent sperm to cross again. But how exactly does this action of ZP2 cause the zona pellucida to harden? The reasons for this remained unclear.

  Now, new research published in the journal Cell offers a new explanation, revealing how ZP2 cleavage prevents multiple sperm fertilisation by altering the overall structure of the zona pellucida - contrary to conventional wisdom, the system does not prevent sperm from binding to the zona pellucida, but rather acts as a "gateway" by preventing sperm from crossing the zona pellucida. The "gatekeeper" role.

  A joint team of researchers began by combining X-ray crystallography and cryo-electron microscopy to investigate the three-dimensional structure of the ZP protein. We know that the two ends of a protein are the nitrogen-containing amino terminus (N-terminus) and the carbon-containing carboxyl terminus (C-terminus). For ZP family proteins, the functional diversity of different members depends on the structure of their N-terminal region (NTR), which contains different numbers and kinds of protein structural domains.

  It was found that cleavage of ZP2 allows the N-terminal region to oligomerise after fertilisation and interact with other molecules to form a highly stable homotetramer. This interaction forms a cross-linked network that makes these ZP proteins more tightly connected. In this way, the "mesh" of ZP proteins in the zona pellucida is tightened like a zip tie, preventing additional sperm from penetrating the zona pellucida.

  In addition, the research team also predicted the structure of the human zona pellucida in conjunction with AlphaFold, revealing the interconnection of two protofibrils composed of ZP proteins, which form a left-handed double helix. The N-terminal region of one of the ZP1/ZP2/ZP4 subunits stands out from it. Together, these results explain the mechanism by which ZP2 oligomerisation leads to the hardening of the zona pellucida, thereby preventing sperm from crossing.

  It is worth noting that previous studies have suggested that the ZP2-N1 structural domain in the N-terminal region of the ZP2 subunit is indispensable as a receptor when spermatozoa bind to the zona pellucida. This study, however, found that in the absence of ZP2-N1 expression, sperm were still able to bind to the zona pellucida, although few sperm could actually penetrate the zona pellucida. This suggests that ZP2-N1 is not a necessary receptor for sperm binding, but that this structural domain plays other key roles for fertility. A new question that arises is what is the real receptor on the zona pellucida? The team plans to explore this further.

  Overall, this discovery explains the structural changes in the zona pellucida after fertilisation from a molecular perspective, which also has important implications for the future of reproductive medicine. Changes in the zona pellucida after fertilisation are crucial for female fertility, as it ensures that the developing embryo is protected before implantation in the uterus. Therefore, this new discovery not only provides an explanation for female infertility, but may also help in the development of non-hormonal contraceptives.