Studies on the modification of ram spermatozoa by ejaculation and cryopreservation and the effects of Binder of Sperm Proteins Taylor Pini A thesis submitted to the Faculty of Science, The University of Sydney in fulfilment of the requirements for the degree of Doctor of Philosophy © 2018 ii Declaration Apart from the assistance mentioned in the acknowledgements, the studies contained within this thesis were planned, executed, analysed and written by the author, and have not been previously submitted for any degree to a University or institution. Taylor Pini BAnVetBioSc (Hons I) iii Acknowledgements No great work is accomplished alone, and this thesis is a great reflection of that. Firstly I must thank the animals involved, who tirelessly provided samples, never complained and always kept my passion for research alive. To my supervisors and fall-back parents Simon and Tamara, I don’t know where I would be without you. Your support and encouragement has helped me over hurdles I couldn’t have faced alone and has always pushed me to be the best I can be. Your comedic relief has been the light in the darkness at times. I’m so grateful to have had you as mentors, and hope you will both continue to be a sounding board for me throughout my career. There are many other people to thank, especially the extended ARGUS family, past and present - thank you for the stimulating hallway chats, answering my random questions, providing a well needed break from writing and making lab meetings and social events so fun. A special mention to Kim, my technical support angel, who for four years was my number one ‘go to’ for just about any question I had. To my lab buddies, Jess, Jessie, Naomi, Kelsey, Emmah, Reina and Kiri; this couldn’t have happened without your efforts, which were often superhuman and always offered freely. You are all wonderful friends and I salute your commitment to science, often over sleep, your significant others, and a social life. Thank you also to Cam, the king of 2 o’clock trivia and an unwavering “yes” to an afternoon visit to the pub. Thank you to Byron, Keith and Cameron for being our tireless sheep people, transporting rams back and forth and always being on hand for a day of semen collection. Thanks also to Southern Meats abattoir in Goulburn, and particularly David and Teresa, both for allowing me on site to collect testes and ovaries, and for somehow making that a very enjoyable experience. I can’t say I miss the very early alarms and drives to Goulburn and back, but I will miss our regular catch ups. Proteomics was a scary field to even contemplate; I have to thank iv the Sydney Mass Spectrometry staff Ben Crossett, Angela Connolly and David Maltby for their patient and unwavering guidance in all things mass spec. One of the best experiences throughout my candidature was the pleasure of working with an international contingent. To Xavier, Guillaume, Clement, Valerie, Ana Paula, Lucie and all of the wonderful scientists from the reproductive physiology and mass spec facilities at INRA, thank you for your hard work in producing our proteomic data and crafting beautifully purified BSPs. Thank you to Xavier especially, for introducing me to the finer things in life (aged sheep cheese) and fostering such a lovely, ongoing connection between our research groups. I also must extend my appreciation to Bart; your visits are always a source of great pleasure, many good stories and exciting new hypotheses. Your advice has always been worth its weight in gold. To my friends, many of whom were completing their PhDs at the same time as me, thank you for being so understanding, agreeing that sometimes there is no silver lining and always being there to listen. To my parents, Pauline and Joe, I know you were proud of me already, but I’ve done you one better and gotten a doctorate. Thanks to you and our extended family for your ongoing love and support, and for giving me all the opportunities that brought me to this point. Finally, to the two special guys in my life, Dylan and Atlas, thank you for your patience, encouragement and belief in me. v Summary Frozen thawed ram spermatozoa must be deposited directly into the uterus via laparoscopy as they are unable to traverse the cervix in sufficient numbers to achieve acceptable fertility. Seminal plasma has been heralded as a possible solution to this problem, as it appears to enhance sperm cervical transit. However, the mechanism by which seminal plasma, or indeed cryopreservation, alters spermatozoa to subsequently influence cervical transit is unknown. This thesis explores how seminal plasma and cryopreservation affect the structure and function of ram spermatozoa, with the ultimate aim of improving the in vivo performance of cryopreserved ram semen. Results reported herein showed seminal plasma exposure and cryopreservation significantly altered the sperm proteome and glycocalyx, and interactions between spermatozoa and immune cells. Seminal plasma added two unique proteins (LEG1, EDIL3) and significantly increased the abundance of 39 proteins, including the Binder of Sperm Proteins (BSPs) 1 and 5. Ejaculated ram spermatozoa showed significant protein conservation (95%) compared to sperm proteomes from the human, bull, stallion, rooster and trout. Seminal plasma exposure significantly decreased available sialic acid, and increased N-acetylglucosamine on the sperm surface. Seminal plasma also protected spermatozoa from immune cells, significantly reducing the percentage of neutrophils bound to ejaculated spermatozoa (by 67.5%). However, it did not prevent opsonin mediated binding. Egg yolk in freezing media was shown to contribute 15 proteins to ram spermatozoa, including vitellogenins, apolipoproteins and complement C3. Cryopreservation itself increased the abundance of 27 proteins (e.g. SERPINB1, FER) and decreased 24 proteins (e.g. TOM1L1, CSN1G2). Chaperones constituted 20% of the proteins lost following freezing, suggesting important functional consequences. Cryopreservation also reduced available galactose and N- acetylglucosamine and increased available mannose on the sperm surface. Freezing induced sugar changes were related to the modification, loss or gain of glycoproteins. The effects of cryopreservation on immune cell interaction were unclear due to inhibition of neutrophil binding by vi cryodiluent. This was not due to egg yolk, which significantly enhanced binding (by 29.0%). Further investigation is required to determine whether cryopreservation itself alters phagocytic susceptibility of spermatozoa. The physiological and potential cryoprotective effects of BSPs were investigated, given their high abundance in seminal plasma and demonstrated transfer to the sperm surface during ejaculation. Under significant cAMP upregulation, BSP1 showed both pro- and de-capacitating effects on fresh spermatozoa, including promotion of cholesterol efflux and the acrosome reaction, and limitation of membrane lipid disorder and protein tyrosine phosphorylation respectively. In contrast, BSP5 had limited effects on capacitation related processes. Pre freeze supplementation with BSPs improved post thaw motility (by 36.2%) and sperm kinematic parameters in an artificial mucus medium. Pre freeze supplementation with either BSP1 or BSP5 significantly improved post thaw progressive motility (by 15.6% and 15.0% respectively) and viability (by 11.2% and 10.4% respectively), and decreased freezing induced tyrosine phosphorylation. Supplementation with BSPs did not minimise loss of acrosome integrity or membrane lipid disorder. Further work is necessary to confirm translation of these positive outcomes to improvements in vivo. The findings of this thesis demonstrate that the physiological process of ejaculation and the artificial practice of cryopreservation significantly alter the molecular profile and function of spermatozoa. Seminal plasma exposure confers additional proteins, alters the sperm glycocalyx, protects spermatozoa from immune cells and regulates the timing of capacitation. Cryopreservation also alters the sperm proteome and glycocalyx, possibly increasing susceptibility of this sperm type to dynamic environmental pressures within the female reproductive tract. Further investigation of the degree to which BSPs benefit ram spermatozoa in the female tract is warranted given their significant effects on sperm cryosurvival and physiology demonstrated herein. The importance of this protein family in ram sperm physiology offers a potential avenue for future improvements in the fertility of frozen thawed ram spermatozoa following cervical insemination. vii Table of Contents DECLARATION .................................................................................................................... II ACKNOWLEDGEMENTS .................................................................................................... III SUMMARY ........................................................................................................................... V LIST OF ABBREVIATIONS ................................................................................................ XII LIST OF TABLES .............................................................................................................. XV LIST OF FIGURES ........................................................................................................... XVI LIST OF PUBLICATIONS................................................................................................ XXII 1. REVIEW OF THE LITERATURE .....................................................................................