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Proteomic Identification and Evolutionary Analysis of Primate Reproductive Proteins

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Proteomic Identification and Evolutionary Analysis of Primate Reproductive Proteins Proteomic identification and evolutionary analysis of primate reproductive proteins Katrina G. Claw A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Washington 2013 Reading Committee: Willie J. Swanson, Chair Michael J. MacCoss, Chair Deborah A. Nickerson Program Authorized to Offer Degree: Genome Sciences ©Copyright 2013 Katrina G. Claw University of Washington Abstract Proteomic identification and evolutionary analysis of primate reproductive proteins Katrina G. Claw Chair of the Supervisory Committee: Associate Professor Willie J. Swanson and Associate Professor Michael J. MacCoss Genome Sciences Sex and reproduction have long been recognized as drivers of distinct evolutionary phenotypes. Studying the evolution and molecular variation of reproductive proteins can provide insights into how primates have evolved and adapted due to sexual pressures. In this dissertation, I explore the long-term evolution of reproductive proteins in human and non-human primates. I first describe the evolutionary diversification of sperm and eggs, and what drives them to diverge. I then describe rapidly evolving proteins in the egg and in sperm-egg interactions. I then present the use of a unique combination of genomic and proteomic technologies to study the evolution of seminal fluid proteins. With proteomics, I identify and quantify the abundance of a large proportion of uncharacterized seminal fluid proteins from 8 primate species with diverse mating systems. Using evolutionary analyses, I find rapidly evolving seminal fluid proteins and candidates genes with evolutionary rates and protein abundances that are correlated with mating system variation. I then explore the phylogentic relationships between putatively coevolving sperm-egg fusion genes. I find evidence that two pairs of sperm-egg fusion genes have correlated evolutionary rates along primate phylogenetic branches, which may indicate that they are genetically interacting. I conclude by discussing how to disentangle the selective pressures causing reproductive protein divergence, the value of using proteomics in comparative evolutionary studies, and future directions for identifying interacting fertilization proteins. Table of Contents Chapter 1: Rapid Evolution in Eggs and Sperm ....................................................................... 1 Introduction................................................................................................................................................... 1 Diversity of Sperm and Egg.......................................................................................................................... 1 Sperm–Egg Interactions................................................................................................................................ 8 Evolutionary Hypotheses ............................................................................................................................ 11 Chapter 2: Evolution of the Egg: New Findings and Challenges ........................................... 24 Introduction................................................................................................................................................. 24 Overview of fertilization process................................................................................................................ 25 Basic Egg Structure..................................................................................................................................... 26 The cumulus oophorus............................................................................................................................ 27 The Zona Pellucida................................................................................................................................. 30 The Plasma Membrane........................................................................................................................... 36 Rapid Evolution of Egg proteins................................................................................................................. 37 ZP2 and ZP3 on the Zona Pellucida....................................................................................................... 39 CD9 on the plasma membrane ............................................................................................................... 39 Mechanisms to limit polyspermy................................................................................................................ 39 Conclusion .................................................................................................................................................. 41 Chapter 3: Comparative proteomics and evolution of primate seminal fluid proteins ....... 46 Introduction................................................................................................................................................. 46 Materials and Methods................................................................................................................................ 48 Primate Samples ..................................................................................................................................... 48 Sample preparation and Mass Spectrometry.......................................................................................... 49 Normalization and quantification of relative protein abundance .......................................................... 51 Coding sequences and multiple sequence alignments............................................................................ 52 Evolutionary analysis ............................................................................................................................. 52 Evolutionary correlation ........................................................................................................................ 53 Gene Loss................................................................................................................................................ 54 Gene Ontology analysis.......................................................................................................................... 54 Results/Discussion ...................................................................................................................................... 55 Seminal fluid protein composition and gene ontology ........................................................................... 55 Protein abundance within and between species ..................................................................................... 56 Seminal fluid proteins are subject to rapid evolution............................................................................. 59 Is there a correlation between evolutionary rates and mating systems?................................................ 60 Pseudogenization in seminal fluid proteins............................................................................................ 61 Conclusion .................................................................................................................................................. 62 Chapter 4: Detecting coevolution in mammalian sperm-egg fusion proteins ....................... 74 Introduction................................................................................................................................................. 74 Methods....................................................................................................................................................... 76 Sequencing and identification of sites under positive selection ............................................................. 76 Correlation of evolutionary rates to predict interacting proteins.......................................................... 77 Results......................................................................................................................................................... 79 Evolutionary analysis of sperm-egg fusion genes .................................................................................. 79 Coevolution between sperm-egg fusion genes........................................................................................ 80 Discussion ................................................................................................................................................... 81 Chapter 5: Conclusions and Future Directions ....................................................................... 89 Detecting the causes behind rapidly diverging reproductive proteins ........................................................ 90 i Using proteomics for comparative studies.................................................................................................. 91 Future directions for functionally verifying interacting reproductive proteins........................................... 92 References.................................................................................................................................... 94 ii List of Figures Figure 1. Sperm and egg morphological diversity………………………………………..... 14 Figure 2. The fruit fly Drosophila bifurca…………………………………………………..15 Figure 3. Detecting positive selection……………………………………………………… 16 Figure 4. Abalone population distribution…………………………………………………. 17 Figure 5. Primate mating systems………………………………………………………….. 18 Figure 6.
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