Hard or Die: Molecular Principles of Zona pellucida hardening

• Applicant: Dr. Dirk Fahrenkamp, Ph.D.
• Subject Area: Reproductive Medicine, Urology ; Structural Biology ; Cell Biology
• Term: from 2018 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 401763100

To give rise to a new individual, sperm has to penetrate the zona pellucida (ZP), an extracellular glycoprotein matrix surrounding mammalian oocytes. After fertilization, the ZP undergoes extensive remodelling in a process known as ZP hardening. Hardening of the zona is characterized by a number of correlating observations including decreased ZP elasticity, ZP thinning, increased resistance against proteolytic digestion and loss of sperm binding - processes whose molecular principles are essentially unknown but are believed to be related to cleavage of the egg coat protein and sperm receptor ZP2. Through this, ZP hardening protects the developing embryo from environmental hazards and establishes a definitive block to polyspermic fertilization.Human infertility has been estimated to affect 48.5 million couples in 2010, though the cause of infertility often remains elusive. To treat infertility, assisted reproductive techniques (ART) are regularly applied that, however, yield a baby-take-home rate of only 5-29%. Premature ZP hardening of human oocytes during in vitro culture represents a major problem lowering the success rates of ART. However, advances in inhibiting unintended premature ZP hardening are impeded by the fact that ZP hardening mechanisms are poorly understood. My project aims to identify how processing of ZP2 regulates ZP hardening and sperm binding at fertilization. ZP2 cleavage occurs in its second domain (ZP2-N2) and is thought to be mediated by the protease Ovastacin, which is released by oocytes following gamete fusion. However, ZP2 structures are so far only available for the sperm-binding (ZP2-N1) and the ZP-C domain. Therefore, I propose to produce recombinant ZP2 containing at least the first two domains and Ovastacin to cleave ZP2 in vitro. By solving the structures of both, ZP2 and cleaved ZP2 (ZP2f), I want to define the molecular events that trigger ZP hardening and inactivate sperm receptor activity.Moreover, recombinant ZP2 and ZP2f will be subjected to interaction studies to screen for novel interactions of ZP2f with either itself, intact ZP2 or the other egg coat proteins that would provide a rationale to explain ZP hardening. ZP protein complexes will be functionally characterized by mutagenesis experiments and structure determination will be carried out by means of X-ray crystallography, or, if feasible, cryo-EM to conclusively characterize ZP2 cleavage-induced protein cross-links that mediate ZP hardening. To understand how ZP2 cleavage transforms the global architecture of the ZP, I want to use focus ion beam milling (FIB) to produce ultrathin ZP slices, which, in turn, can be subjected to cryoelectron tomography to obtain structural information of native ZP filaments with a near-to-molecule resolution.By clarifying how ZP2 processing regulates ZP hardening and abolishes sperm binding to the ZP, this project will provide important insights into the molecular basis of fertilization in mammals.

• DFG Programme: Research Fellowships
• International Connection: Sweden
• Host: Professor Dr. Luca Jovine