Hard or Die: Molecular Principles of Zona pellucida hardening

为了产生一个新的个体，精子必须穿透透明带（ZP），这是一种围绕在哺乳动物卵母细胞周围的细胞外糖蛋白基质。受精后，ZP在一个称为ZP硬化的过程中经历了广泛的重塑。带硬化的特征是一系列相关的观察结果，包括ZP弹性降低、ZP变薄、抗蛋白水解消化能力增强和精子结合丧失——这些过程的分子原理基本上是未知的，但被认为与卵膜蛋白和精子受体ZP2的切割有关。通过这种方式，ZP硬化保护发育中的胚胎免受环境危害，并对多精受精建立了明确的阻碍。据估计，2010年人类不孕症影响了4850万对夫妇，尽管导致不孕症的原因往往难以捉摸。为了治疗不孕症，经常使用辅助生殖技术（ART），然而，将婴儿带回家的比率仅为5-29%。在体外培养过程中，人类卵母细胞过早的ZP硬化是降低ART成功率的主要问题。然而，抑制非预期的ZP过早硬化的进展受到ZP硬化机制知之甚少这一事实的阻碍。我的项目旨在确定ZP2的加工如何调节受精时的ZP硬化和精子结合。ZP2的切割发生在它的第二个结构域（ZP2- n2），被认为是由蛋白酶Ovastacin介导的，该蛋白酶在配子融合后由卵母细胞释放。然而，迄今为止，ZP2结构仅适用于精子结合（ZP2- n1）和ZP-C结构域。因此，我建议制备至少含有前两个结构域和Ovastacin的重组ZP2，在体外切割ZP2。通过解析ZP2和裂解的ZP2 （ZP2f）的结构，我想定义触发ZP硬化并使精子受体活性失活的分子事件。此外，重组ZP2和ZP2f将进行相互作用研究，以筛选ZP2f与自身、完整的ZP2或其他鸡蛋外壳蛋白的新相互作用，这将为解释ZP硬化提供理论依据。ZP蛋白复合物将通过诱变实验进行功能表征，结构测定将通过x射线晶体学进行，或者，如果可行的话，冷冻电镜来最终表征介导ZP硬化的ZP2切割诱导的蛋白质交联。为了了解ZP2解理如何改变ZP的整体结构，我想使用焦点离子束铣削（FIB）来生产超薄的ZP片，而超薄的ZP片又可以进行低温电子断层扫描，以接近分子分辨率获得天然ZP细丝的结构信息。通过阐明ZP2加工如何调节ZP硬化并消除精子与ZP的结合，该项目将为哺乳动物受精的分子基础提供重要见解。

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.