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Cilia- and Flagella-Associated Protein 69 Regulates Olfactory Transduction Kinetics in Mice

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Cilia- and Flagella-Associated Protein 69 Regulates Olfactory Transduction Kinetics in Mice The role of CFAP69 in the male reproductive system by Frederick N. Dong A dissertation submitted to Johns Hopkins University in conformity with the requirements of the degree of Doctor of Philosophy Baltimore, Maryland December, 2018 Abstract The importance of reproduction for life, human or otherwise, cannot be overstated: without reproduction, an organism and, eventually, its species cease to exist. Thus, infertility is a devastating condition. Shockingly, however, nearly 15% of human couples struggle with infertility, with male infertility contributing to about half of these cases. Furthermore, the cause of half of male infertility cases still cannot be identified. About 30% of male infertility cases are believed to have a genetic component, but many of the genes required for male fertility and what these gene products contribute are still undiscovered. In this dissertation, we set forth the first studies of the role of cila- and flagella-associated protein 69 (CFAP69) in spermatogenesis. In Chapter 1, I briefly review spermatogenesis and some of its known defects. In Chapter 2, we demonstrate that Cfap69 is expressed in germ cells in the mouse testis and find that CFAP69 is essential for both human and mouse fertility. Its absence preserves the overall progression of spermatogenesis but causes impaired spermiogenesis and disorganization of sperm flagellum components, thus leading to multiple morphological abnormalities of the flagella and infertility. In Chapter 3, we examine CFAP69 itself. We examine the prediction that CFAP69 contains an armadillo repeat domain and the implication that it interacts with other proteins. However, we find that this protein is largely insoluble under physiological extraction conditions not only in heterologous expression systems, but also in mouse testis and sperm, suggesting that the insolubility reflects the function of CFAP69. Finally, in Chapter 4, we explore the implications of our results for human health, the utility of CFAP69 in further studying spermatogenesis at a molecular level, improved tools for studying difficult proteins like CFAP69, and the mysterious roles of CFAP69 in biological systems as diverse as olfaction and platelet morphology. Advisor: Haiqing Zhao, Ph.D. Second Reader: Xin Chen, Ph.D. Committee Members: Allan Spradling, Ph.D. William Wright, Ph.D. ii Table of Contents Abstract ...................................................................................................................................... ii Table of Contents ...................................................................................................................... iii List of Tables .............................................................................................................................. iv List of Figures ............................................................................................................................. v Chapter 1: Introduction .............................................................................................................. 1 1-1. The importance of reproduction ............................................................................................ 2 1-2. Modes of reproduction .......................................................................................................... 3 1-3. Spermatogenesis and its defects ............................................................................................ 5 Chapter 2: Absence of CFAP69 causes male infertility due to multiple morphological abnormalities of the flagella in humans and mouse ................................................................ 12 2-1. Introduction ......................................................................................................................... 13 2-2. Results .................................................................................................................................. 14 2-3. Discussion ............................................................................................................................ 33 2-4. Methods ............................................................................................................................... 37 2-5. Supplemental Data ............................................................................................................... 45 Chapter 3: Studying the mechanism of CFAP69 function .......................................................... 56 3-1. Introduction ......................................................................................................................... 57 3-2. Results .................................................................................................................................. 59 3-3. Discussion ............................................................................................................................ 70 3-4. Methods ............................................................................................................................... 73 Chapter 4: Concluding remarks ................................................................................................ 79 4-1. CFAP69 and human health ................................................................................................... 80 4-2. Towards a molecular-level understanding of spermiogenesis .............................................. 82 4-3. Studying problematic proteins ............................................................................................ 84 4-4. One CFAP69 function or many? ............................................................................................ 85 Appendix I. Cilia- and Flagella-Associated Protein 69 Regulates Olfactory Transduction Kinetics in Mice ........................................................................................................................ 87 Appendix II. Lamin B1 is reQuired for mature neuron-specific gene expression during olfactory sensory neuron differentiation ............................................................................................... 100 Bibliography ........................................................................................................................... 114 iii List of Tables Table 2-1. Detailed semen parameters in the two MMAF individuals harboring a CFAP69 mutation. ................................................................................................................................. 32 Table S2-1. ................................................................................................................................ 53 Table S2-2. Primer sequences used for Sanger sequencing verification of CFAP69 variations and respective melting temperatures (Tm). ............................................................................. 55 Table S2-3. Primers used for RT-qPCR of CFAP69 in human. .................................................... 55 Table S2-4. Primer sequences used in human CFAP69 RT-PCR and respective melting temperatures (Tm). .................................................................................................................. 55 Table S2-5. All CFAP69 (C7orf63) variations identified by WES. ............................................... 55 Table 3-1. The 50 most abundant proteins detected by mass spectrometry after pulldown from mouse testis using recombinant CFAP69. ........................................................................ 69 iv List of Figures Figure 2-1. Morphology of normal and CFAP69 mutant spermatozoa, and the mutations identified in CFAP69-mutant individuals .................................................................................. 22 Figure 2-2. CFAP69 immunostaining in human spermatozoa from controls and CFAP69 mutant individuals. .................................................................................................................. 23 Figure 2-3. SPAG6 and SPEF2 immunostainings are affected by mutations in CFAP69. ........... 24 Figure 2-4. EXpression of CFAP69 in the mouse testis. ............................................................. 26 Figure 2-5. Scanning electron microscopy analysis of sperm from epididymides of wildtype and Cfap69 KO mice. ................................................................................................................ 29 Figure 2-6. The progression of spermatogenesis in Cfap69 KO mice is preserved, but flagellum components are disorganized. ................................................................................................. 31 Figure S2-1. Relative mRNA EXpression of human CFAP69 transcripts. .................................... 47 Figure S2-2. Electropherograms of Sanger sequencing for the two CFAP69-mutated individuals compared to reference sequence. ............................................................................................ 48 Figure S2-3. RT-PCR analyses on peripheral whole blood cells from individual CFAP69_1 showing mRNA decay. .............................................................................................................. 49 Figure S2-4. AXonemal inner and outer dynein arms are not affected by the absence of CFAP69. .................................................................................................................................... 50 Figure S2-5. Normal acrosome development and nuclear elongation can be observed in testes of Cfap69 KO mice. ..................................................................................................................
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