Development and Application of Structural Tools to Investigate Mammalian Gametes

Abstract

Mammalian fertilization is a highly complex biological process that relies on the coordinated interaction of two specialized reproductive cells, sperm and oocytes (eggs). Understanding the structural and molecular basis of how these gametes function and interact is essential for addressing infertility and improving assisted reproductive technologies (ART). In this thesis, I investigated mammalian gamete biology and the dynamic cellular changes involved in fertilization using advanced imaging techniques, primarily cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET). These methods enabled high-resolution visualization of reproductive cells in a near-native state, revealing critical insights into key biological processes essential for successful fertilization. The first chapter provides a historical and technical overview of the role of electron microscopy in reproductive biology. It outlines the limitations of conventional EM approaches and highlights how recent advances in cryo-EM and cryo-ET have transformed our ability to visualize cellular architecture at molecular resolution. We reviewed landmark discoveries in sperm and oocyte ultrastructure, and discussed how new methodologies are opening doors to long-standing questions in fertilization research. In the subsequent chapters, we explored the architecture and functional specialization of mammalian sperm. Using cryo-ET, we analysed sperm from pigs, horses, and mice, revealing both conserved and species-specific structural features. The discovery of microtubule inner proteins (MIPs) provides new understanding of how the flagellum maintains flexibility and mechanical stability during movement. Additionally, we characterized structural differences in the proximal and distal centrioles, suggesting distinct roles in sperm motility and early embryonic development. Another key finding was the identification of conserved supramolecular protein arrays linking mitochondria to the cytoskeleton, supporting the metabolic and structural demands of motile sperm. The fourth chapter focuses on acrosomal exocytosis, a key step in sperm capacitation. Our cryo-ET analysis revealed a stepwise disassembly of the acrosomal matrix and extensive membrane remodelling, offering new insights into how sperm prepare for and execute egg fusion. In the fifth chapter, I present a structural and biochemical analysis of the bovine zona pellucida (ZP), the extracellular matrix surrounding the oocyte and a key regulator of sperm binding. We identified dynamic structural changes in the ZP across different stages of reproduction, highlighting its active role in fertilization. A major achievement of this work is the determination of the first high-resolution structure of the zona pellucida from any mammalian species. These findings significantly enhance our understanding of how the egg coat mediates fertilization and provide a foundation for future structural studies of mammalian egg coats. Finally, I developed and optimized a novel cryo-ET sample preparation workflow tailored for large-volume specimens, enabling in situ imaging of mammalian oocytes. This approach overcomes longstanding technical challenges related to oocyte size and shape and lays the groundwork for future studies of egg biology and fertilization in a native cellular context and can be adapted to other large biological systems, such as tissues and organoids. Together, this thesis provides a comprehensive structural framework for understanding mammalian gamete function and fertilization. By bridging molecular detail with cellular organization, this work advances our fundamental knowledge of reproductive biology and offers new directions for improving infertility diagnostics and assisted reproductive technologies.