Physiological and Molecular Consequences of Large Y Chromosome Long Arm Deletions in Mice Emma Elizabeth Philippa Johnson MRes Emmanuel College Department of Pathology University of Cambridge September 2017 This dissertation is submitted for the degree of Doctor of Philosophy. This dissertation is submitted to the University of Cambridge in accordance with requirements of the degree of Doctor of Philosophy in the School of the Biological Sciences. Page 1 of 331 Page 2 of 331 Physiological and Molecular Consequences of Large Y Chromosome Long Arm Deletions in Mice Emma Elizabeth Philippa Johnson Summary The mammalian Y chromosome contains genes important for male sexual maturity and reproduction. The mouse Y chromosome long arm harbours a number of multi-copy genes whose absence or reduced representation has been linked to sperm defects and offspring sex ratio distortion in favour of females. Understanding the biological mechanisms of sex ratio distortion and related sperm aberrations could not only result in benefits for fertility research, but also in the development of methods for large scale animal breeding pre-implantation sex selection. The distortion has been linked to an intragenomic conflict between the X and Y chromosomes that impacts spermiogenesis. Since the proportion of X- and Y-bearing sperm does not differ in affected animals, and there is no selective destruction of male embryos post-fertilisation, a functional difference must exist between the X- and Y-bearing sperm. This thesis describes the investigation into the physiological and molecular mechanisms of a large Y-chromosome long arm deletion in the mouse model MF1XYRIIIqdel. The examination of physiological characteristics revealed a distinct sperm morphology within the deletion model. Detailed characterisation of sperm shape demonstrated that aberrations consistently occur within specific regions of the sperm head, linking the distorted morphology to particular maturation stages in the sperm cycle. Using sperm fluorescence in situ hybridisation, a novel and detailed comparison of X- and Y- bearing sperm has shown that a subtle distinction in shape also exists between the X- and Y- bearing sperm in the deletion model. Breeding data were examined and showed a skew towards female offspring and a slightly reduced litter size. Sperm enzyme activity assays did not reveal altered hyaluronidase activity in MF1XYRIIIqdel sperm. Physiological differences between X- and Y- bearing sperm must result from differential gene expression, complicated by the syncitial nature of sperm development. To explore this, a detailed molecular characterisation of the MF1XYRIIIqdel phenotype in developing haploid spermatids was performed. Cellular elutriation and fractionation techniques were employed to separate spermatids at different stages of maturation and isolate different subcellular compartments. Differences in the transcriptional profile between these populations were analysed by microarray and RNA sequencing analysis of total and micro RNA. This work yielded a collection of coding and non-coding transcripts which show distinctive expression and compartmentalisation differences between the deletion model and its wild-type counterpart across several sperm maturation stages. Combining these strategies has led to the identification of several gene products potentially implicated in observed physiological differences and the offspring sex ratio skew, providing candidate genes for further research. Page 3 of 331 Page 4 of 331 Author’s Declaration This dissertation is the result of my own work conducted between 2013 and 2017, and includes nothing which is the outcome of work done in collaboration except as specified in the text. I further state that no substantial part of my dissertation has been submitted, or, is being concurrently submitted for any such degree, diploma or other qualification at the University of Cambridge or any other University or similar institution. This thesis does not exceed the prescribed word limit of 60,000 words set by the School of Biological Sciences, University of Cambridge. Emma Elizabeth Philippa Johnson MRes Page 5 of 331 Page 6 of 331 This thesis is dedicated to my family and friends. Thank you for everything. Page 7 of 331 Page 8 of 331 Acknowledgements This thesis, and the experience I have had conducting the work summarised in it, have only come about because of the involvement of several people. I want to start by thanking my supervisor, Professor Nabeel Affara; a man whom, like a great work of art, excels at comforting the disturbed and disturbing the comfortable. Nabeel has made me feel welcome in the lab from the moment I arrived and has made it harder for me to say goodbye. The best of what I have learnt from Nabeel has been all that we’ve spoken about away from the bench. I only hope to find another mentor from whom I can gain as much. Thank you to my second supervisor, Dr Peter Ellis, who is now based at the University of Kent. Peter is an exceptionally pleasant man to know, whose detailed knowledge of this (and nearly every) subject has entertained me from the beginning. I am pleased to say that he only appears to use his powers for good. Thank you, Peter, for always being on the other end of an email. My third supervisor, Dr Alan Mileham, has supported me in things I’ve undertaken and made sure that opportunities I have had have been the best they can be. He has provided excellent conversation on the occasions that we have had the pleasure of meeting and has been a great source of advice. My final lab mentor is Dr Benjamin Skinner. Although not an official supervisor, Ben is the person I have worked most closely with over the past three years. Ben is unflagging in his enthusiasm for science. Thank you for the constant stream of insults and for taking mine well in return. You have been a great source of help and guidance over the past few years and I am grateful for it. I will not miss your puns. I would like to thank Jo Bacon and Kim Lachani for their kind tuition, especially at the beginning of my time in the lab, and for their efforts and assistance with several experiments that did not quite make it into the thesis. Thank you also to Jo, and to Kerry Harvey for the kind words they provided when I needed them most. Thank you Dr Carole Sargeant for sharing some of the fascinating things she knows over the last few years and for thoroughly enriching my time in the lab. Thank you to fellow PhD student Courtney Landers for being a provider of much needed lunch time walks and musings. Thank you to Claire for always a cheery ‘hello’ and to all members of the lab for making the last four years what they have been. A final thank you to our wonderful part II students Myrto Vlazaki and Becki Nunn, who are both inspiring and lovely people. From the Cambridge Genomics Services team, I’d first like to thank Dr Ed Farnell for answering all (or most of) my long list of questions with enthusiasm and for his questionable sense of humour. Thank you especially to Julien Bauer and Maria Gomez for being the foundation of my understanding of bioinformatics. I am very grateful for all the time they spent with me answering my many questions and helping me when I needed it. Thank you also to Dr Alex Covney, Dr Emily Clemente, Phil Howden, Chris Reitter and Marta Andrada for all their help and delightful conversation; and to Phil and Chris for answering my early morning phone calls to the lab! Thanks to Carl Wignall for Page 9 of 331 responding humorously to my IT woes and providing thoughtful “constructive” criticism of my handling of technical issues. My sincerest thanks go to the kind members of the Griffin lab, and in particular to Dr Claudia Rathje for not only allowing me to use their lab space, but setting me up and booking time slots for me on their excellent microscope. Thank you for being my second lab-home. Additional thanks must go to Claudia and Bill (and the parrots) for housing me and entertaining me during a number of my visits to Canterbury. Colleges can be a big part of student life at Cambridge, and Emmanuel College has been an important part of mine. I would like to thank the Emmanuel porters in particular. Their help and kindness has been indispensible, and they have always made me feel welcome whenever I visit the college, no matter how long I’ve been away. From further afield, I must mention Jessie Martin and Laramie Pence. During my time at the University of Maryland in the summer of 2016, both Jessie and Laramie did everything they could to share their skills and knowledge with me and, most importantly, they made my time there thoroughly memorable. My gratitude extends to all members of the Telugu lab, who were hugely accommodating, facilitated my first experience of microinjection and CRISPR based experiments, and helped expand my understanding of the field. There are many more people I would like to thank, and without taking up several more pages, I will not be able to do so in full. So, I’d instead like to briefly thank my mentor Dr Paolo D’Avino, Dr Debbie Drage, Dr Nigel Slater, the Pathology graduates, and my friends at Emmanuel College and beyond. I’d also like to thank my sponsors and funders, the BBSRC, Genus Plc, and KTN for all the experiences and opportunities they have enabled and supported. It would not have been possible without them. I have been extremely lucky to have enjoyed so much of what I have done over the years and this all begins with having remarkable parents. Thank you for everything you have done and still do for me.