University of Dundee MASTER OF SCIENCE Investigating the clinical value of hyperactivation and intracellular calcium signalling of human spermatozoa and their value in drug screening Ruso, Halil Award date: 2013 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: 01. Oct. 2021 MASTER OF SCIENCE Investigating the clinical value of hyperactivation and intracellular calcium signalling of human spermatozoa and their value in drug screening Halil Ruso 2014 University of Dundee Conditions for Use and Duplication Copyright of this work belongs to the author unless otherwise identified in the body of the thesis. It is permitted to use and duplicate this work only for personal and non-commercial research, study or criticism/review. You must obtain prior written consent from the author for any other use. Any quotation from this thesis must be acknowledged using the normal academic conventions. It is not permitted to supply the whole or part of this thesis to any other person or to post the same on any website or other online location without the prior written consent of the author. Contact the Discovery team ([email protected]) with any queries about the use or acknowledgement of this work. Investigating the Clinical Value of Hyperactivation and Intracellular Calcium Signaling of Human Spermatozoa and Their Value in Drug Screening Halil Ruso MSc by Research University of Dundee December 2013 ii Table of Contents List of Figures ............................................................................................................... iv List of Tables .................................................................................................................. x Declaration .................................................................................................................... xi Acknowledgements ....................................................................................................... xii Summary ..................................................................................................................... xiii List of Abbreviations .................................................................................................... xv Chapter 1: General Introduction ............................................................................. 1 1.1 Sperm Structure .................................................................................................... 1 1.1.1 Sperm Head .............................................................................................................. 2 1.1.2 Sperm Flagellum ....................................................................................................... 2 1.2 Control of Sperm Motility ..................................................................................... 2 1.2.1 Flagellum .................................................................................................................. 3 1.2.2 cAMP control of motility .......................................................................................... 5 1.3 Capacitation .......................................................................................................... 9 1.3.1 Molecular basis of capacitation ............................................................................. 10 1.3.2 Calcium and capacitation ....................................................................................... 13 1.4 Hyperactivation ................................................................................................... 15 1.4.1 Definition and importance of hyperactivation ........................................................ 15 1.4.2 Function of hyperactivation .................................................................................... 16 1.4.3 Control of hyperactivation ...................................................................................... 18 1.4.4 Physiological inducers of hyperactivation ............................................................. 23 1.5 Infertility ............................................................................................................. 24 1.5.1 Definition, prognosis, diagnosis, and treatment of male infertility ........................ 24 1.6 Aims and objectives ............................................................................................. 27 Chapter 2 : Investigation of Human Sperm Hyperactivation Levels and Intracellular Calcium Signalling and Their Relation with Fertilization Rates ...... 28 2.1 Introduction ............................................................................................................ 28 2.2 Materials and Methods ........................................................................................... 30 2.2.1 Experimental Design .................................................................................................. 30 2.2.2 Media and Chemicals................................................................................................. 30 2.2.3 Sperm Preparation ..................................................................................................... 31 2.2.4 Evaluation of Hyperactivation ................................................................................... 33 2.2.5 Intracellular Calcium Detection ................................................................................ 35 2.2.6 Definition of defective hyperactivation and Ca2+ response ....................................... 36 2.2.7 Fertilization Rates ...................................................................................................... 36 2.2.8 Statistical Analysis ..................................................................................................... 36 2.3 Results .................................................................................................................... 39 2.3.1 Hyperactivation Levels and Efficacy of the Drugs .................................................... 39 2.3.2 Hyperactivation Levels of IVF Patients and Relationship with Fertilization Rates .. 41 2.3.3 Calcium Signalling Induction with Agonists in IVF Patients and Donors ................ 46 2.3.4 Hyperactivation and Intracellular Ca2+ Responses of ICSI Patients ......................... 48 2.3.5 Defective Hyperactivation and Intracellular Ca2+ Responses of Different Populations 50 2.4 Discussion ............................................................................................................... 51 iii Chapter 3 : Effect of Phosphodiesterase Inhibitors on Human Sperm Motility and Calcium Signalling ................................................................................................ 55 3.1 Introduction ............................................................................................................ 55 3.2 Materials and Methods ........................................................................................... 57 3.2.1 Experimental design ................................................................................................... 57 3.2.2 Media and Chemicals................................................................................................. 57 3.2.3 Sperm Preparation ..................................................................................................... 57 3.2.4 Phosphodiesterase Inhibition ..................................................................................... 58 3.2.5 Evaluation of Hyperactivation ................................................................................... 58 3.2.6 Intracellular Calcium Detection ................................................................................ 58 3.2.7 Statistics ..................................................................................................................... 59 3.3 Results .................................................................................................................... 60 3.3.1 Effect of PDE Inhibitors on Motility Parameters ...................................................... 60 3.3.2 Effect of PDE Inhibitors on Calcium Signalling and Its Relation with Motility ........ 66 3.4 Discussion ............................................................................................................... 77 Chapter 4 : Screening a Chemogenomics Library Using Calcium as a Surrogate . 80 4.1 Introduction ............................................................................................................ 80 4.2 Materials and Methods ........................................................................................... 82 4.2.1 Experimental design ................................................................................................... 82 4.2.2