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Molecular and Physical Interactions of Human Sperm with Female

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Molecular and Physical Interactions of Human Sperm with Female MOLECULAR AND PHYSICAL INTERACTIONS OF HUMAN SPERM WITH FEMALE TRACT SECRETIONS by Asma M Hamad A thesis submitted to The University of Birmingham For the degree of DOCTOR OF PHILOSOPHY College of Medical and Dental Sciences School of Clinical and Experimental Medicine Institute of Metabolism and Science Research University of Birmingham May 2017 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. ABSTRACT To achieve fertilisation, human sperm have to navigate and interact with the female reproductive tract (FRT) on molecular and mechanical levels. The current knowledge of some aspects of both types of interactions are limited and they were examined in this research. Proteomic analysis of crude and depleted human follicular fluid (hFF) by three proteomic approaches identified 479 hFF-proteins of which 22% were novel. A table of hFF-proteins, compiled from twenty-four hFF proteomic studies, resulted in 1586 hFF proteins; a resource for folliculogenesis and discovery of hFF biomarkers. A comparative proteomic study of media-capacitated human sperm versus capacitated sperm in the presence of hFF revealed certain hFF proteins were acquired by sperm during capacitation. Comparative metabolomics revealed some elevated metabolites in the sperm capacitation-media following 6-hour incubation compared to 1-hour swim-up, which may have relevance to sperm energy metabolism and potentially to sperm signalling mechanisms in the FRT causing remodelling for fertilisation and preparation for implantation. Sperm micro-particle image-velocimetry revealed an average fluid velocity around the motile sperm of ≤ 25- 45 µm/s and ≤ 20-35 µm/s in low and high viscosity media respectively. The averaged fluid vorticity manifested a trail of spatially confined mixing of the fluid surroundings motile sperm. To the best of our knowledge, this work is the first to conduct a comparative proteomic analysis of human sperm versus sperm-hFF interaction and to perform metabolomics analysis of human sperm capacitation media at two time points. The novel perspective of mechanical aspects of sperm motility by studying fluid I velocity and vorticity around motile sperm adds a new approach to the study of sperm motility and chemotaxis. In conclusion, sperm-FRT interactions involve complex molecular and physical interactions and regulatory events. Further research of these interactions may enhance our understanding of potential applications for improved assisted reproductive techniques’ outcome and possible diagnostic approaches of infertility. II Dedications To the memory of my father, I miss you every second of my life To the memory of my brothers Mustafa and Abdullah, I hope you are in heaven To my devoted mother, I love you so much To my sister Abier, you taught me the love of books To all my sisters, Eman, Hana, Nada, Nisreen and Raja, I love you all so much To my brothers, Ahmed and Osama, I hope you all the best in your life III Acknowledgments I would like to thank my lead supervisor Dr. Jackson Kirkman-Brown for his profound support and advice throughout my PhD. I am deeply grateful to his guidance and unconditional support. I would like to thank Dr. Linda Lefievre, my co-supervisor, for her great input and unlimited support throughout the years and hope her all the best. I also would like to thank Dr. Sarah Conner for her valuable feedback and input and I really appreciate her kind support. I am also grateful to Dr. Ashley Martin and Dr. Douglas Ward for running the mass spectrometry analyses of my proteomic data and their input for the experimental designs and proteomic approaches applied in this research. I am also deeply grateful to Dr. Hermes Gadelha (University of York) and Dr. David Smith for their kind collaboration and development of the MATLAB codes for my sperm micro-particle image velocimetry’s data analysis and their fruitful discussions and valuable feedback. I am also extremely grateful to Dr. Warwick Dunn for running the mass spectrometry analysis of my metabolomics data, metabolomics data annotation and comparative analysis of the data and his valuable discussions and feedback. My immense gratitude goes to Dr. Lorraine Frew for the provision of research sperm donors throughout my PhD study and her kind cooperation and I would like to thank all the donors and patients at the Assisted Conception Unit, Birmingham Women’s Hospital who made this work possible. I am also highly grateful to Mrs Sarah Magson who facilitated my meetings with my supervisors and enabled smooth and organised PhD course and conferences’ attendance throughout my PhD study. I would like to extremely thank my colleague Benjamin Taiwo for his valuable discussions and feedback and I really appreciate his friendship. I am also grateful to his brother Dami, who I never met, for his kind support. I also would like to thank the Libyan Ministry of IV Higher Education for funding my PhD and the staff at the Libyan Cultural Attaché in London with special thanks to my supervisors Dr. Ali Al Faitori and Fowzi Alshawish for the precious administrative assistance. Finally I am very grateful to my family in Libya for their unconditional love and support and to my husband Ahmed for his great support and my children Mohamed and Suhaib who I hope will, one day, have their own PhD degree. V TABLE OF CONTENTS Chapter 1: GENERAL INTRODUCTION ....................................................................... 1 1.1 The Sperm cell ........................................................................................................ 2 1.1.1 Spermatogenesis: ............................................................................................. 2 1.1.2 Sperm cell structure: ........................................................................................ 4 1.1.3 Sperm maturation in the Epididymis: .............................................................. 7 1.1.4 Semen ............................................................................................................ 10 1.2 The female reproductive tract (FRT): ................................................................... 13 1.2.1 Menstrual cycle: ............................................................................................ 13 1.2.2 Oogenesis: ..................................................................................................... 16 1.2.3 Folliculogenesis: ............................................................................................ 17 1.3 Sperm journey toward the oocyte: ........................................................................ 28 1.3.1 Sperm transport through the vagina and cervix: ............................................... 28 1.3.2 Sperm transport through the uterus and uterotubal junction: ............................ 29 1.4 Regulation of sperm function ............................................................................... 33 1.4.1 Sperm capacitation: ....................................................................................... 33 The biphasic role of Ca2+ in capacitation: .................................................................... 35 Tyrosine phosphorylation in sperm capacitation: ........................................................ 36 The role of de-capacitation factors in regulation of capacitation: ................................ 38 1.4.2 Post-translational modifications (PTM) of sperm proteins : ......................... 39 1.4.3 Sperm motility ............................................................................................... 40 1.4.4 Sperm guidance mechanisms in the FRT ...................................................... 42 1.4.5 Acrosome reaction (AR):............................................................................... 44 1.5 FF and sperm function and kinetics: ..................................................................... 48 1.5.1 FF and sperm capacitation: ............................................................................ 49 1.5.2 FF and sperm motility: .................................................................................. 49 VI 1.5.3 FF and sperm chemotaxis .............................................................................. 51 1.5.4 FF and sperm acrosome reaction: .................................................................. 52 1.5.5 FF and sperm quality: .................................................................................... 55 1.6 Techniques: ........................................................................................................... 55 1.7 Research aims: ...................................................................................................... 64 Chapter 2: IDENTIFICATION OF NOVEL HUMAN FOLLICULAR FLUID PROTEINS BY PROTEOMIC TECHNIQUES ............................................................ 66 2.0 Abstract ................................................................................................................. 67 2.1 Introduction: .........................................................................................................
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