Swanson, Willie J.Carlisle, Jolie Anna2022-01-262022-01-262021Carlisle_washington_0250E_23715.pdfhttp://hdl.handle.net/1773/48265Thesis (Ph.D.)--University of Washington, 2021Reproductive proteins mediating fertilization commonly exhibit rapid sequence diversification driven by positive selection. This pattern has been observed among nearly all taxonomic groups, including mammals, invertebrates, and plants, and is remarkable given the essential nature of the molecular interactions mediating fertilization. In chapters two and three of my thesis, I discuss how variation in reproductive gene content between species can be generated through subfunctionalization or gene loss. In chapter four, I discuss how sequence diversification of gamete recognition proteins can create boundaries to hybridization. The marine gastropod mollusk abalone (genus Haliotis) is a classic model for fertilization. Its two acrosomal proteins (lysin and sp18) are ancient gene duplicates with unique gamete recognition functions. Through detailed genomic and bioinformatic analyses we show how duplication events followed by sequence diversification has played an ongoing role in the evolution of abalone acrosomal proteins. The common ancestor of abalone had four members of its acrosomal protein family in a tandem gene array that repeatedly experienced positive selection. Further, a more recent species-specific duplication of both lysin and sp18 in the European abalone H. tuberculata is described. We hypothesize that, in a manner analogous to host/pathogen evolution, sperm proteins are selected for increased diversity through extensive sequence divergence and recurrent duplication driven by conflict mechanisms. The abalone lysin-VERL interaction is a classic model for understanding the molecular mechanisms mediating egg coat dissolution. During abalone fertilization, sperm lysin dissolves the vitelline envelope of the egg in a species-specific manner by binding VERL ZP-N domains. Using surface plasmon resonance, a quantitative biophysical method, we have measured the binding affinities between heterospecific and conspecific lysin-VERL domain pairs. In our study, we discovered that species-specific binding between lysin and VERL ZP-N Repeat 1 (VR1) was consistent with the experimentally measured species-specific VE dissolution function of lysin. ZP2, a mammalian egg coat protein, shows similarity in evolutionary patterns, protein structure, and function to abalone VERL despite extensive divergence between the proteins. Experimentally guided molecular docking indicates that both abalone and mammalian ZP-N domains bind the same region of lysin, consistent with a shared mechanism between mammalian and abalone sperm-egg interactions. My thesis explores how the diversification of fertilization genes in sequence and gene content across even closely related species can impact mechanisms of fertilization. Further, my thesis emphasizes the value of using diverse model systems for investigating mechanisms of fertilization.application/pdfen-USCC BY-NC-NDabaloneevolutionfertilizationmolecular biologymolecular evolutionreproductive biologyGeneticsEvolution & developmentGeneticsThesis