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Short Abstinence: Impact on the Seminal Plasma Proteome and Accessory Sex Gland Secretions Thesis Presented in Fulfilment Of

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Short Abstinence: Impact on the Seminal Plasma Proteome and Accessory Sex Gland Secretions Thesis Presented in Fulfilment Of Short abstinence: Impact on the seminal plasma proteome and accessory sex gland secretions By Dale Mark Goss Thesis presented in fulfilment of the requirements for the degree of Master of Science in the Faculty of Medicine and Health Sciences at Stellenbosch University Supervisor: Professor Stefan S. Du Plessis Co-supervisor: Professor Gerhard van der Horst Stellenbosch University https://scholar.sun.ac.za Declaration By submitting this dissertation, I declare that the entirety of the work contained therein is my own, original work, that I am the sole author thereof, that reproduction and publication thereof by Stellenbosch University will not infringe any third party rights and that I have not previously in its entirety or in part submitted it for obtaining any qualification. Dale M. Goss March 2018 Copyright © 2018 Stellenbosch University All rights reserved i Stellenbosch University https://scholar.sun.ac.za Abstract Various studies have sought to determine the typical versus optimal abstinence period after which semen samples should be collected, and many have been found to be contradictory. Several factors influence the semen microenvironment and subsequent sperm basic and functional parameters. In this study we focused on important biomarkers of prostate, seminal vesicle and epididymal secretion. Furthermore, extensive proteomic analysis of seminal plasma was performed for a more comprehensive understanding of the observations. Semen samples were obtained from normozoospermic donors (n = 16) after 4 days and 4 hours of ejaculatory abstinence (EA) and standard semen analysis was performed with the assistance of computer aided sperm analysis (CASA), while the seminal plasma citric acid, neutral alpha- glucosidase and fructose concentrations were measured photometrically with commercially available assay kits. Protein identification was performed using advanced mass spectrometric techniques and pathways were analysed on both Reactome and String databases. Results of this study displayed significant decreases in semen volume, sperm concentration, total sperm count, and pH after 4 hours of ejaculatory abstinence when compared to 4 days. Furthermore, increases in total sperm motility and progressive motility after short EA periods were observed, accompanied by significant reductions in all epididymal and accessory sex gland biomarker concentrations. However, due to the decreased sperm number, these concentrations translated to a significant increase in citric acid and a decrease in fructose available per spermatozoon, which, along with the effects on semen pH, could more than likely be responsible for increased metabolic function and subsequent increase in the available energy for sperm motility. Proteomic analysis identified 22 differentially expressed seminal plasma proteins, with 18 proteins upregulated after 4 days of EA and 3 upregulated after 4 hours of EA. Of the proteins differentially expressed, 5 proteins from 4 days of EA and 2 proteins from 4 hours of EA, were identified to be related to sperm function and thus selected for pathway analysis. These ii Stellenbosch University https://scholar.sun.ac.za proteins were found to be prominent components of cellular processes including metabolism, apoptosis and cell process regulation, which can be linked directly and indirectly to sperm motility parameters. To conclude, the mechanisms by which very short EA improves sperm motility depends on seminal plasma composition thereby affecting metabolic function and protein interactions. iii Stellenbosch University https://scholar.sun.ac.za Opsomming Verskeie studies het probeer om die tipiese versus optimale onthoudingsperiode te bepaal waarna semenmonsters versamel moet word, maar heelwat teenstrydighede is gevind. Verskeie faktore beïnvloed die semen mikro-omgewing en daaropvolgende sperm-basiese en funksionele parameters. In die huidige studie is daar gefokus op belangrike biomerkers in sekresies afkomstig vanaf die prostaat, seminale vesikels en epididimis. Uitgebreide proteomiese analises is ook op die seminale plasma uitgevoer om sodoende 'n meer omvattende begrip vir die waarnemings te verkry. Semenmonsters is bekom vanaf normozoospermiese skenkers (n = 16) na 4 dae en 4 ure van ejakulatoriese onthouding (EO) en ʼn standaard semenanalise is uitgevoer met behulp van rekenaargesteunde spermanalise (RGSA), terwyl die seminale plasma sitroensuur, neutrale alfa-glukosidase en fruktose konsentrasies fotometries bepaal is met kommersieel beskikbare toetsstelle. Proteïenidentifikasie is uitgevoer met behulp van gevorderde massa spektrometriese tegnieke en proteoompad interaksies is geanaliseer op beide Reactome en String databasisse. Die resultate van hierdie studie toon beduidende afnames in semenvolume, spermkonsentrasie, totale spermtelling en pH aan ná 4 uur van EO in vergelyking met 4 dae. Verder is toenames in totale spermmotiliteit en progressiewe motiliteit na kort EO periodes waargeneem. Dit is vergesel van 'n betekenisvolle vermindering in alle epididimale en bykomende geslagsklierbiomerkerkonsentrasies. Nietemin, as gevolg van die verminderde spermgetal, herlei hierdie konsentrasies egter tot 'n beduidende toename in sitroensuur en 'n afname in fruktose beskikbaar per spermatozoon, wat tesame met die effekte op semen pH waarskynlik verantwoordelik kan wees vir verhoogde metaboliese funksie en daaropvolgende toename in die beskikbare energie vir spermmotiliteit. Proteomiese analise het 22 differensiaal uitgedrukte seminale plasma proteïene identifiseer, waarvan 18 proteïene opgereguleer was na 4 dae van EO en 3 opgereguleer was na 4 ure van EO. Van die proteïene wat differensiaal uitgedruk was, is 5 proteïene vanaf 4 dae EO en iv Stellenbosch University https://scholar.sun.ac.za 2 proteïene vanaf 4 ure EO gekoppel aan spermfunksie en gekies vir verdere proteoompad interaksie analise. Daar is bevind dat hierdie proteïne prominente rolspelers is in sellulêre prosesse, insluitende metabolisme, sellulêre motiliteit, apoptose en selprosesregulering. Om mee af te sluit, die meganismes waardeur baie kort EO spermmotiliteit verbeter berus op seminale plasma samestelling met gevolglike effekte op metaboliese funksie en proteïen interaksies. v Stellenbosch University https://scholar.sun.ac.za Acknowledgements Professor Stefan Du Plessis, Emeritus Professor Gerhard van der Horst and Bashir Ayad, for their academic insight, unwavering support and greatly appreciated optimism. Dr Marè Vlok, (Central Analytical Facility) and Dr Suzelle Hattingh (Division of Medical Physiology) for their expertise guidance in proteomics. Division of Medical Physiology, in particular the Stellenbosch University Reproductive Research Group, for all the assistance and many happy memories. Stellenbosch University for granting me a bursary allowing me to pursue this degree. vi Stellenbosch University https://scholar.sun.ac.za Dedication To my parents Mark and Zelna, and my partner Donna, for your endless support and love, without which I would not have been able to achieve what I have. vii Stellenbosch University https://scholar.sun.ac.za Table of Contents Page Declaration i Abstract ii Opsomming iv Acknowledgements v Dedication vi Table of Contents vii List of Tables xi List of Figures xii Abbreviations xiv viii Stellenbosch University https://scholar.sun.ac.za Chapter 1: Introduction 1.1. Background 1 1.2. Motivation for study 2 1.3. Preliminary study 3 1.4. Thesis organisation 5 1.5. Setting of the study 6 1.6. Aims and objectives 6 1.7. Conclusion 6 Chapter 2: Literature review 2.1. Abstinence and fertility 8 2.2. Spermatogenesis, sperm capacitation and motility 10 2.3. Important seminal plasma components 14 2.4. Proteomics 18 Chapter 3: Materials and methods 3.1. Ethical clearance 23 3.2. Basic semen analysis 23 3.2.1. Volunteers 23 3.2.2. Semen sample collection 23 3.2.3. Motility 24 3.2.4. Viability 24 3.2.5. Storage of samples 25 3.3. Epididymal and accessory sex gland biomarkers 25 3.3.1. Citric acid assay 26 3.3.2. Fructose assay 26 3.3.3. Episcreen plusTM – Neutral alpha-glucosidase assay 27 3.4. Statistical analysis 28 ix Stellenbosch University https://scholar.sun.ac.za 3.5. Proteomic analysis 28 3.5.1. Sample preparation and SDS-PAGE 28 3.5.2. In-gel digestion 29 3.5.3. Liquid chromatography 30 3.5.4. Mass spectrometry 30 3.5.5. Bioinformatics 31 Chapter 4: Results 4.1. Objective 1: Basic and functional semen analysis 33 4.2. Objective 2: Epididymal and accessory sex gland biomarker assays 37 4.3. Objective 3: Proteomics 38 Chapter 5: Discussion 5.1. Objective 1 43 5.2. Objective 2 47 5.3. Objective 3 52 5.3.1. Proteins upregulated after 4 days of EA 52 5.3.1.1. Heat-shock protein A2 53 5.3.1.2. 26S proteasome non-ATPase subunit 8 54 5.3.1.3. 14 kDa phosphohistidine phosphatase 59 5.3.1.4. Human Phosphatidylethanolamine-binding protein 4 60 5.3.1.5. 2,4-dienoyl-CoA reductase, mitochondrial 62 5.3.2. Proteins upregulated after 4 hours of EA 64 5.3.2.1. Calcium-binding protein 39 64 5.3.2.2. Deoxyribonuclease I 69 5.4. Limitations of the study/recommendations 70 Chapter 6: Conclusion 71 x Stellenbosch University https://scholar.sun.ac.za Bibliography 75 Appendices Appendix A: Epididymal and accessory sex gland secretion optical densities 90 Appendix B: Manuscript sent in to peer-reviewed journal 95 Appendix C: Proteomic analysis results 111 xi Stellenbosch University https://scholar.sun.ac.za List of Tables Page Table 4.1: Representative summary of the differences observed amongst the macro- and microscopic semen and spermatozoa parameters
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