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Preclinical Evaluation of TEX101 Protein As a Male Infertility Biomarker and Identification of Its Functional Interactome

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Preclinical Evaluation of TEX101 Protein As a Male Infertility Biomarker and Identification of Its Functional Interactome Preclinical Evaluation of TEX101 Protein as a Male Infertility Biomarker and Identification of its Functional Interactome by Christina Schiza A thesis submitted in conformity with the requirements for the degree of Doctorate of Philosophy Laboratory Medicine and Pathobiology University of Toronto © Copyright by Christina Schiza 2017 Preclinical Evaluation of TEX101 Protein as a Male Infertility Biomarker and Identification of its Functional Interactome Christina Schiza Doctor of Philosophy Laboratory Medicine and Pathobiology University of Toronto 2017 Abstract TEX101 is a testis-specific cell surface protein expressed exclusively in the male germ cells, and it was previously suggested as a biomarker for the differential diagnosis of male infertility. Molecular function of TEX101 is not known, but previous studies demonstrated that murine TEX101 was a cell membrane chaperone, essential for fertilization. In this work, we employed quantitative proteomic strategies to investigate the clinical and the functional aspects of human TEX101 protein. To facilitate translation of TEX101 into clinic, we first developed and optimized an immunoassay for TEX101 measurement in biological fluids. We then evaluated the performance of TEX101 ELISA in 805 seminal plasma samples of fertile, sub-fertile, and infertile men. We demonstrated the clinical utility of TEX101 to evaluate vasectomy success, to stratify azoospermia forms and subtypes, and to predict the success of sperm retrieval in patients diagnosed with non-obstructive azoospermia. To gain insights into human TEX101 function, we employed mass spectrometry approaches to investigate the physical and the transient TEX101 interactome. Co-immunoprecipitation-mass spectrometry revealed a testis-specific complex TEX101-DPEP3 which was validated by hybrid immunoassay in testicular tissues and spermatozoa. In addition, we discovered a TEX101 rs35033974 homozygous knockdown model, which carried a missense variation leading to near- ii complete protein degradation. We then performed global proteomic analysis of TEX101 knockdown spermatozoa, and we identified the transient interactome of TEX101. New knowledge on TEX101 will provide insights into reproductive biology, facilitate better diagnostics of male infertility, contribute to rational selection of assisted reproduction treatments, and offer new protein targets to develop non-hormonal male contraceptives. iii Acknowledgments Every ending is a new beginning! The past five years have been a new experience for me. It was a challenge and a bet with myself. This thesis is my reward. There have been many people to guide me and to share my excitement and my disappointment throughout these years. First, I would like to thank my supervisor, Dr. Eleftherios P. Diamandis, for giving me the opportunity to join ACDC Lab, and to pursue a PhD in one of the top universities worldwide. Dr. Diamandis’ challenging approach made me realize my inner strength, and gave me the power to fight and not give up. I would also like to acknowledge the members of my advisory committee, Dr. Keith Jarvi and Dr. Alexander Romaschin, for their valuable advice throughout my PhD. A very special thanks to Dr. Jarvi for his positive energy, and for providing all the biological specimens for my studies. In addition, I would like to acknowledge the members of my final oral examination committee, Dr. G. Schmitt-Ulms, Dr. V. Kulasingam, and Dr. R. Oko for reviewing my thesis and for providing comments and feedback. I would also want to thank Dr. Harry Elsholtz for the support and aid for all academic needs. I wish to express my appreciation to two great scientists and labmates, Dr. Andrei P. Drabovich and Dr. Dimitrios Kormpakis. I was very lucky to work closely with both of them throughout my PhD. They were always willing to share their knowledge and experience, and they tought me how to focus on my goals, and to be patient. They encouraged me to keep trying improving my skills, and they were a great example that hard work is always rewarded. I would also like to thank Andrei for reviewing my thesis and my manuscripts. This journey would have been lonely if I did not have my labmates and friends next to me. Lampros, Ilijana, and Dimitris are the people that I shared moments of excitement, disappointment, stress, endless complaining about PhD life, but also fun by exploring new things in Toronto. A special thanks to Ihor Batruch for trying his best to keep our instruments alive and in good shape, but most of all for being Ihor! Finally, a special thanks to our lab manager Antoninus for keeping the lab in balance for so many years, and to Susan Lau for recruiting all the patients, and for the administrative support. iv Some 8,000 km away, but always next to me, my family and my friends. I would like to thank Christina, Myrto, Konstantina, Georgia and Cleo for being my anchors back home, and for believing in me. Special thanks to Cleo for all the technical support, and for staying up late to keep me company while I was writing my thesis. Last but not least, I want to thank my parents, Giorgos and Anastasia, for their unconditional support and all the sacrifices they made, to give me and my sister a better future. I have to acknowledge that although it was really hard for them to have both their children away, they never doubted about our choices. Finally, I would like to thank my sister. Although she is younger than me, she has always been an example of determination, strength, and courage. v Table of Contents Abstract .......................................................................................................................................... ii Acknowledgments ........................................................................................................................ iv Table of Contents ......................................................................................................................... vi List of Tables ............................................................................................................................... xii List of Figures ............................................................................................................................. xiii List of Appendices ........................................................................................................................xv List of Abbreviations ................................................................................................................ xvii 1 Introduction ...............................................................................................................................2 1.1 Infertility .............................................................................................................................2 1.2 The Male Reproductive System ........................................................................................3 1.2.1 Anatomy and Physiology of the Male Reproductive System ..................................4 1.2.1.1 Testes and spermatogenesis .......................................................................4 1.2.1.2 Genital ducts and sperm maturation ..........................................................6 1.2.1.3 Accessory glands and production of seminal fluid ....................................7 1.2.2 Sperm transport and maturation ...............................................................................7 1.3 Male Infertility ...................................................................................................................9 1.3.1 Subtypes of male infertility ......................................................................................9 1.3.2 Diagnosis of male infertility ..................................................................................10 1.3.3 Current treatments ..................................................................................................12 1.3.4 Emerging male infertility biomarkers ....................................................................13 1.4 Innovative proteomics strategies to study clinical and functional aspects of male reproduction and infertility ............................................................................................15 1.4.1 Reagents and tools to study testis-specific proteins ...............................................15 1.4.2 Protein-protein interactions ....................................................................................16 1.4.3 Global and targeted mass spectrometry approaches ..............................................17 1.5 TEX101 protein: a biomarker of spermatogenesis and male infertility .....................18 1.5.1 TEX101 expression pattern at transcript and protein level ....................................18 1.5.2 TEX101 localization during spermatogenesis and sperm maturation ...................20 1.5.3 Suggested functional roles of TEX101 protein ......................................................21 vi 1.5.4 TEX101 as a biomarker for the differential diagnosis of azoospermia .................23 1.6 Rationale and Objectives of the present study ..............................................................27 1.6.1 Rationale ................................................................................................................27 1.6.2 Hypothesis..............................................................................................................28 1.6.3 Objectives ..............................................................................................................29
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