University of Dundee MASTER OF SCIENCE Novel Modulators of Human Sperm Motility King, Louise Award date: 2014 Link to publication General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 26. 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Contact the Discovery team ([email protected]) with any queries about the use or acknowledgement of this work. ‘Novel Modulators of Human Sperm Motility’ Louise Victoria King MSc (by research) Reproductive Biology University of Dundee May 2014 2 Table of Contents List of Figures and Tables ............................................................................................................. 6 Acronyms and Abbreviations ....................................................................................................... 8 Acknowledgements ..................................................................................................................... 10 Declaration .................................................................................................................................. 10 Summary ..................................................................................................................................... 11 CHAPTER 1 General Introduction ............................................................................................. 12 1.1 Male infertility .................................................................................................................. 13 1.2 Diagnosis of male infertility ............................................................................................. 13 1.2.1 Semen analysis ........................................................................................................... 13 1.2.2 Sperm Function Assays .............................................................................................. 14 1.3 Asthenozoospermia ........................................................................................................... 15 1.4 The Use of ART ................................................................................................................ 15 1.5 Capacitation ...................................................................................................................... 16 1.6 Initiation of sperm motility ............................................................................................... 17 1.6.1 Hyperactivation .......................................................................................................... 18 1.7 The role of Ca2+ signalling in sperm motility ................................................................... 21 1.7.1 Calcium channels in sperm ........................................................................................ 21 1.7.2 Calcium stores in sperm ............................................................................................. 27 1.8 Flexstation screening assay ............................................................................................... 28 1.9 Chemical stimulation of sperm motility ............................................................................ 29 1.10 Computer assisted sperm analysis (CASA) of sperm motility ........................................ 30 1.11 Thesis Aims .................................................................................................................... 32 CHAPTER 2 Methods and Materials .......................................................................................... 34 2.1 Reagents and Stocks ......................................................................................................... 35 2.2 Media used for donor samples .......................................................................................... 36 2.3 Samples ............................................................................................................................. 36 2.3.1 Ethical Approval ........................................................................................................ 36 2.3.2 Semen Samples .......................................................................................................... 37 2.4 Sperm preparation ............................................................................................................. 38 2.5 CASA parameters adopted in all experiments .................................................................. 38 2.6 Gradient experiment (Chapter 3) to determine most suitable fraction to be used as a surrogate for patients ............................................................................................................... 39 2.7 Assessment of calcium and motility induced by novel compounds ................................. 39 2.7.1 Flexstation Assay Screen (experiments conducted by Sarah Matins da Silva in combination with the Dundee Drug Discovery Unit (DDU)) ............................................. 39 3 2.7.2 Preparation for motility assessment of novel compounds .......................................... 40 2.7.3 Sperm penetration into artificial viscous medium (Kremer test) ............................... 41 2.8 Assessment of the Acrosome Reaction ............................................................................. 42 2.9 Hypo-osmotic swelling (HOS) test ................................................................................... 43 2.10 Statistical analysis ........................................................................................................... 44 CHAPTER 3 Identification of a sub-group of donor spermatozoa after density gradient centrifugation that is comparable to a patient sample ................................................................. 45 3.1 Introduction ....................................................................................................................... 46 3.2 Results ............................................................................................................................... 48 3.3. Discussion ........................................................................................................................ 53 CHAPTER 4 Development of Standard Operating Procedure for Drug Screening .................... 55 4.1 Introduction ....................................................................................................................... 56 4.1.1 Minimum number of cells counted to reduce sampling error .................................... 57 4.1.2 Variation in motility assessed by different slide types ............................................... 57 4.1.3 Between-donor and within- donor biological variation ............................................. 58 4.1.4 Effect of DMSO on sperm motility ............................................................................ 59 4.2 Results ............................................................................................................................... 60 4.2.1 Development of methods for motility assessment by CASA ..................................... 60 4.2.2 Development of methods for the assessment of motility for drug screening ..................... 65 4.3 Discussion ......................................................................................................................... 69 4.3.1 Repeatability and Reliability of system ..................................................................... 69 CHAPTER 5 Motility Analysis of Sperm treated with Hit Compounds identified through a Flexstation High Throughput Screening Assay .......................................................................... 76 5.1 Introduction ....................................................................................................................... 77 5.1.1 Cannabinoid agonists ................................................................................................. 79 5.1.2. Mitogen activated protein kinases ............................................................................. 81 5.1.3 c-MET kinase inhibitor .............................................................................................