Identification of Phosphoproteins Involved in Sperm

IDENTIFICATION OF PHOSPHOPROTEINS INVOLVED IN SPERM MATURATION AND FERTILITY. A dissertation submitted to Kent State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy by Suranjana Goswami August 2018 © Copyright All rights reserved Except for previously published materials Dissertation written by Suranjana Goswami B.Sc., University of Calcutta, India, 2009 M.Sc., University of Calcutta, India 2011 Ph.D., Kent State University, USA, 2018 Approved by Dr. Srinivasan Vijayaraghavan , Chair, Doctoral Dissertation Committee Dr. Douglas W. Kline , Members, Doctoral Dissertation Committee Dr. Gary Koshi , Dr. Sanjaya Abeysirigunawardena , Dr. Andrew Jasnow , Accepted by Dr. Laura G. Leff , Chair, Department of Biological Sciences Dr. James L. Blank , Dean, College of Arts and Sciences TABLE OF CONTENTS ............................................................................................................... iii LIST OF FIGURES ..........................................................................................................................v LIST OF TABLES ....................................................................................................................... viii ABBREVIATIONS ....................................................................................................................... ix DEDICATION ............................................................................................................................... xi ACKNOWLEDGEMENTS .......................................................................................................... xii CHAPTERS .................................................................................................................................... 1 I: Introduction… ............................................................................................................................. 1 1.1 Anatomy of testis ..................................................................................................... 1 1.2 Spermatogenesis ...................................................................................................... 5 1.3 Spermatozoon Structure .......................................................................................... 7 1.4 Sperm Head ............................................................................................................. 9 1.5 Sperm Flagellum ................................................................................................... 10 1.6 Epididymal sperm maturation and motility initiation .............................................11 1.7 Protein Kinases.......................................................................................................13 1.8 Sperm Protein Kinase A ......................................................................................... 17 1.9 sAC and sperm ....................................................................................................... 18 1.10 Protein Phosphatase ............................................................................................... 20 1.11 Protein phosphatase 1 .............................................................................................21 1.12 The PP1 isoforms PP1γ1 and PP1γ2.......................................................................23 1.13 Role of PP1γ2 in testis ............................................................................................ 24 1.14 PP1γ2 in sperm motility & epididymal sperm maturation .....................................26 iii 1.15 The regulators PPP1R2, PPP1R11 and PPP1R7 in sperm ...................................... 27 1.16 Sperm Gsk3a .............................................................................................. 28 1.17 Phospho-proteome ................................................................................................. 30 1.18 Aims ...................................................................................................................... 33 II: Materials and methods...................................................................................................34 III: Aim 1 ........................................................................................................................... 49 Rationale ........................................................................................................................... 49 Result ................................................................................................................................. 52 Summary ........................................................................................................................... 67 Discussion… ..................................................................................................................... 67 IV: Aim 2 .......................................................................................................................... 69 Rationale ........................................................................................................................... 69 Result ................................................................................................................................ 70 Summary ........................................................................................................................... 78 Rationale ........................................................................................................................... 79 Result ................................................................................................................................ 80 Summary ......................................................................................................................... 111 Discussion ....................................................................................................................... 112 REFERENCES ........................................................................................................................... 116 iv LIST OF FIGURES Figure 1. Structure of testis and seminiferous tubules .................................................................... 2 Figure 2. Spermatogenesis .............................................................................................................. 6 Figure 3. Sperm structure ................................................................................................................ 8 Figure 4. Sperm head .................................................................................................................... 10 Figure 5. Flagellar axoneme ...........................................................................................................11 Figure 6. Structure of epididymis ...................................................................................................12 Figure 7. Structure of Protein kinase .............................................................................................. 15 Figure 8. Activation of PKA ......................................................................................................... 17 Figure 9. Structure of Adenylate cyclase ...................................................................................... 19 Figure 10. PP1γ ............................................................................................................................. 24 Figure 11. Expression of PP1 .......................................................................................................... 25 Figure 12. GSK3 .............................................................................................................................29 Figure 13. Analog sensitive PKA ...................................................................................................52 Figure 14. PNBM tagged PKA substrate shown in western blot .....................................................53 Figure 15. Affinity purification using iodoacetyl beads .................................................................54 Figure 16. Coomassie stained gel of BSA and PPP1R2 ..................................................................57 Figure 17. Western blot and SDS-PAGE of the test sample .......................................................... 60 Figure 18. Localization of MCT2 and basigin ................................................................................ 64 Figure 19. Cloning of Ppp1r36 .......................................................................................................71 Figure 20. Northern blot analysis of PPP1R36 in tissues ............................................................... 72 Figure 21. Ppp1r36 is expressed in two different forms .................................................................73 Figure 22. mRNA analysis of PPP1R36 in developmental testis ................................................... 74 v Figure 23. PPP1R36 isoform characterization................................................................................ 75 Figure 24. Co-localization of PP1g2 and PPP1R36 in morphologically normal mouse spermatozoa .........................................................................................77 Figure 25. Northern blot analysis of PP1γ2 binding partners in tissues ........................................... 81 Figure 26. Ppp1r2 expression in tissues.........................................................................................84

Load more