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SERINE/THREONINE PHOSPHATASES: ROLE IN SPERMATOGENESIS AND SPERM FUNCTION A dissertation submitted to Kent State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy By Tejasvi Dudiki December, 2014 © Copyright All rights reserved Except for previously published materials Dissertation written by Tejasvi Dudiki M.S., Osmania University, Hyderabad, India, 2008 B.S., Nizam College, Osmania University, Hyderabad, India, 2006 Approved by Dr. Srinivasan Vijayaraghavan, Chair, Doctoral Dissertation Committee Dr. Douglas W. Kline, Member, Doctoral Dissertation Committee Dr. Wen- Hai Chou, Member, Doctoral Dissertation Committee Dr. Gary Koski, Member, Doctoral Dissertation Committee Dr. Andrea L. Case, Graduate Faculty Representative Accepted by Dr. Eric Mintz, Chair, Department of Biomedical Sciences Dr. James L. Blank, Dean, College of Arts and Sciences ii TABLE OF CONTENTS List of figures…………………………………………………………………………..................iv List of tables…………………………………………………………………………....................ix Acknowledgements……………………………………………………………………................xii 1. Abstract……………………………………………………………………………............1 2. Introduction……………………………………………………………………………......3 2.1 Testis………………………………………………………………………………3 2.2 Spermatogenesis…………………………………………………………………..4 2.3 Spermatozoon structure…………………………………………………...………7 2.4 Epididymal maturation of spermatozoa and initiation of sperm motility….…….13 2.5 Serine threonine protein phosphatases…………………………………………...19 2.6 Regulation of gene expression during spermatogenesis…………………………28 3. Materials and Methods……………………………………………………....................44 4. Aim-I: Identification of PP2A, determination of its biochemical modulations and its role in sperm maturation.……………….……………………………….…………....………57 4.1 Background and Rationale……………………………………..……………….57 4.2 Results for Aim I (A)……………………………...…………………………….59 4.3 Results for Aim I (B)……...……………………………………………….……74 4.4 Discussion………………………………………………………………………87 iii 5. Aim-II: Can PP1 substitute PP1 in spermatogenesis and sperm function if expressed in germ cells?...................................................................................................................93 5.1 Background and Rationale……………………………………………………….93 5.2 Results for Aim II………………………………………………………………102 5.3 Discussion………………………………………………………………………160 6. Conclusion..………………………………………………………………………..…..169 7. Future perspectives…………………………………………………………………….170 8. Bibliography……………………………………………………………………………174 iv LIST OF FIGURES Figure 2.1. Sequential stages in the process of spermatogenesis………………...……………….5 Figure 2.2. Sperm structure……………………………………………………………………….7 Figure 2.3. Features of sperm head…………………………………………………………...…10 Figure 2.4. Features of sperm tail……………………………………………………………….12 Figure 2.5. Regions of the epididymis……………….…………………………….……………15 Figure 2.6. Classification of serine/threonine phosphatases……………………………….........20 Figure 2.7. Conservation among PP1 catalytic subunits……………………………….……..…23 Figure 2.8. Hormonal control of spermatogenesis ………………………………….…………..30 Figure 2.9. Intrinsic regulators of spermatogenesis……………………………………………..31 Figure 2.10. Comparison of alternately spliced (AS) genes across various tissues……………..37 Figure 4.1. Anti-PP2A-C antibody analysis by western blot……………………………………63 Figure 4.2. Immunoprecipitation of PP2A with purified Anti-PP2A antibody…………….…..64 Figure 4.3. Microcystin pull down of PP2A…………………………………………………….64 Figure 4.4. Detection of PP2A in bovine sperm………………….……………………..………65 Figure 4.5. Methylation and tyrosine phosphorylation status of sperm PP2A………………….67 v Figure 4.6. Microcystin pulldown of sperm PP2A………………………………………….…..68 Figure 4.7. Demethylation of PP2A by alkali treatment……………………………………...…70 Figure 4.8. Phosphatase activity of PP2A……………………………………………………….71 Figure 4.9. PME1 and LCMT1 in sperm………………………………………………………..73 Figure 4.10. Possible mechanism for regulation of PP2A methylation in sperm…………..…...75 Figure 4.11. In vivo demethylation sperm PP2A………………………………. ………....……76 Figure 4.12. Timecourse demethylation of PP2A by L-homocysteine and adenosine treatment of sperm………………………………………………………………………………………..……77 Figure 4.13. Progressive demethylation of PP2A by 5nM OA treatment of bovine caudal sperm……………………………………………………………………………………………..78 Figure 4.14. Computer assisted motility analysis of sperm……………………………………..81 Figure 4.15. Catalytic activity of PP2A following its demethylation by L-homocysteine and adenosine treatment……………………………………………………………………………...82 Figure 4.16. Demethylation of PP2A in Distal caput sperm with L-homocysteine and adenosine…………………………………………………………………………………………83 Figure 4.17. Demethylation of PP2A in hyperactivation induced sperm……………………….84 vi Figure 4.18. Western blot to detect tyrosine phosphorylated proteins in 1mM L-homocysteine and adenosine or 5nM OA treated sperm…………………………………….………………….85 Figure 4.19. Serine phosphorylation of GSK3α/β by treatment that results in demethylation of PP2A…………………………………………………………………………………………..…86 Figure 4.20. Proposed model for PP2A regulation of PP1…………………………………...92 Figure 5.1. Schematic of generation of the two PP1γ isoforms…………………………………94 Figure 5.2. Developmental expression of PP1γ1 and PP1γ2 mRNA……………………………97 Figure 5.3. Design of PP1γ1 Rescue constructs…………………………………………………99 Figure 5.4. Breeding scheme of transgenic mouse lines…………………………...………….101 Figure 5.5. Coomassie stained SDS PAGE of expression and purification of His-PP1 and His- PP1…………………………………………………………………………...………………103 Figure 5.6. Western blot analysis for quantification of PP1 in testis………………………..104 Figure 5.7. Western blot analysis for quantification of PP1 in mouse sperm……………….105 Figure 5.8. Western blot comparing PP1 and its interacting proteins in wild type (+/+) and Ppp1cc +/- testis………………………………………………………………………………..107 Figure 5.9. PP1 and PP1 expression ratio in testis of Ppp1cc +/+ and Ppp1cc +/- mice…108 Figure 5.10. Western blot of PP1 levels in Ppp1cc +/- and Ppp1cc +/- brain…………………109 vii Figure 5.11. Western blot comparing Ppp1cc +/+ and Ppp1cc +/- sperm……………………110 Figure 5.12. Western blot analysis of supernatant and pellet fractions of sperm……...……...112 Figure 5.13. PP1 in Rescue I testis and sperm……………………………………………....114 Figure 5.14. Rescue I sperm DIC………………………………………………………………115 Figure 5.15. Western blot analysis for PP1 levels in Rescue II………………………………..116 Figure 5.16. Rescue II sperm morphology………………………………………………… ….117 Figure 5.17. Western blot analysis for quantification of PP1in Rescue testis…………….…118 Figure 5.18. Western blot analysis of Rescue testis and sperm…………………………...…...120 Figure 5.19. Predicted miRNA target sequences of PP1γ1 mRNA……………………………123 Figure 5.20. Conservation of miR449 and miR34 target sequence……………………………125 Figure 5.21. QPCR expression profile of miR449 and miR34……………………………...…128 Figure 5.22. Design of PP1γ1 Rescue IV construct……………………………………………129 Figure 5.23. Western blot analyses of Rescue IV testes……………………………………….132 Figure 5.24. Comparison of PP1γ1 mRNA and protein levels in Rescue lines…………...…...134 Figure 5.25. Western blot analysis of immuno precipitation………………………………..…135 Figure 5.26. Phosphatase activity of PP1 in testis…………………………………………..…137 viii Figure 5.27. PP1γ levels in sperm…………………………………………………………...…141 Figure 5.28. Sperm DIC……………………………………………………………………..…143 Figure 5.29. Immunocytochemistry of sperm……………………………………………….…145 Figure 5.30. Rescue IV sperm motility parameters……………………………………………148 Figure 5.31. Flagellar beat wave form of Rescue IV mice………………………………..…...149 Figure 5.32. Comparision of phoshatase activity in sperm among Ppp1cc +/- and PP1γ Rescues…………………………………………………………………………………….…...151 Figure 5.33. Western blot analyses of Tg; +/+ and Tg; +/- mice testis...……………………..153 Figure 5.34. PP1γ levels in sperm of Tg; +/- mice ……...…...………………………………..154 Figure 5.35. Phosphatase activity in sperm………………………..………............................155 Figure 5.36. Sperm motility parameters………………………………………....…………….158 Figure 5.37. Flagellar beat wave form of sperm…………………………………………….…159 Figure 5.38. Design for generating PP1γ1 Knock-in mouse model……………………………172 ix List of Tables Table 2.1. Protein phosphatase 1 regulatory subunits in mammals……………………………..25 Table 2.2. Protein phosphatase 1 inhibitor proteins in mammals……………………………….26 Table 2.3. List of testis specific genes…………………………………………………………..33 Table 2.4. List of miRNA in mammals………………………………………………………….40 Table 2.5. Comparison of properties of small non-coding RNA……………………………….42 Table 3.1. List of primary antibodies used for western blot analysis……………………………51 Table 3.2. List of primers used for genotyping PCR and RT-qPCR…………………………….55 Table 4.1. Motility analysis of sperm……………………………………………………..……..80 Table 5.1. Levels of PP1 in testis……………………………………………………………104 Table 5.2. Levels of PP1 in sperm…………………………………………………………...106 Table 5.3. Comparitive phenotypic values of Ppp1cc +/+ and Ppp1cc +/- mice……………..108 Table 5.4. Phenotypes of PP1γ1 Rescue mice lines………………………...………………….138 Table 5.5. Fertility results of PP1γ1 Rescue mice lines…………………………………..……139 Table 5.6. Quantitated levels of PP1γ1 incorporated in Rescue sperm……...…………………141 Table 5.7. Phenotypes of Tg; +/- and Tg; +/+ mice ………………………………………….156 x Table 5.8. Fertility results of Tg; +/- and Tg; +/+ mice lines……..…………..…………….156 xi Acknowledgements My journey of becoming a doctorate is not complete without expressing my sincere gratitude to everyone who has been a part of it and made it a little easier and helped me reach my goals. First and foremost