Biochemical and Functional Characterization of Zonadhesin: a Sperm Protein Potentially Mediating Species-Specific Sperm-Egg Adhe
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BIOCHEMICAL AND FUNCTIONAL CHARACTERIZATION OF ZONADHESIN: A SPERM PROTEIN POTENTIALLY MEDIATING SPECIES-SPECIFIC SPERM-EGG ADHESION DURING FERTILIZATION by MING BI, B.S., M.S. A DISSERTATION IN MEDICAL BIOCHEMISTRY Submitted to the Graduate Faculty of Texas Tech University Health Sciences Center Ln Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Advisory Committee Daniel M. Hardy (Chairperson) Beverly S. ChUton Charles H. Faust S. Sridhara Simon C. Williams Accepted AssociateTDean of the Graduate School of Biomedical Sciences Texas Tech University Health Sciences Center May, 2002 © 2002, Ming Bi ACKNOWLEDGMENTS This major accomplishment in my life was made possible by numerous supports from many people who helped me in many different ways. First of all, I would like to thank my mentor Dr. Daniel Hardy. I have learned a lot from him in last five years. He taught me not only academic knowledge and lab techniques in my research field, but also the way to think wisely and to maintain a correct scientific attitude. In addition, his confidence based on his broad knowledge always made me feel comfortable and encouraged during my down time. Beside his great help in study and research, he also helped me to overcome the culture shock and showed great patience in dealing with my poor English. My thanks are also due to all other members in my committee, Dr. Chilton, Dr. Faust, Dr. Sridhara, and Dr. Williams. They gave me lots of valuable suggestions for my research project, and also helped me to keep on track of my study and research plan. I also want to express my special thanks to Dr. Faust. As a graduate advisor for Medical Biochemistry program, he always explained clearly to me about the requirement and procedure on course selection and graduation plan. Furthermore, I would never forget his warm greet in the airport on my first day in Lubbock. My acknowledgements also go to my colleagues in the lab, especially John Hickox. John has helped me a lot in daily lab work, and he has also done significant work in zonadhesin research. I want to thank Tony Cheung. It was Tony who showed me around and taught me lab techniques when I first joined the lab. I also want to thank my other colleagues, Nagesh Uppuluri, Michael Wassler, Steve Tardif, and Joshua Games. They always lent me a hand any time when I needed. Furthermore, 1 would like to thank all other faculty members, staff, and graduate students in my department, who have helped me here and there. I would like to acknowledge the great work from our collaborators. First, I want to thank Dr. Gary Olson and Virginia Winfrey, of Vanderbilt University, for their excellent work on ultrastmctural localization of zonadhesin on spermatozoa using immunoelectron microscopy. I also want to thank Dr. Scott Whisnant, Dr. Heidi Brady, Dr. Andy Herring, Nancy Haden, and Kelly Breazeale of the Department of Animal Science, Texas Tech University for kindly providing animal semen, and personnel in Texas Tech University Farm for continuous supply of pig semen. Finally, I would like to thank my family. It is their continuous support that made me get this far. I would first thank my parents. They have sacrificed so much to support me to complete a series of education goals, financially and spiritually. I will always feel regret that I could not make it to see my mother before she passed away. It was her final advice and inspiration that kept me to stick to my academic plan and to complete my goal today. I also owe a great gratitude to my wife, Xin. Without her continuous unselfish support, it is impossible for me to finish my goal. I really appreciate her hard work in taking care of our son and having a flill-time job at the same time. I would thank my lovely son, Kevin. Making him proud of me is the strongest motivation for me to do my best to complete my goal. To Xin and Kevin. ni TABLE OF CONTENTS ACKNOWLEDGEMENTS ii ABSTRACT x LIST OF FIGURES xii LIST OF ABBREVIATIONS xv CHAPTER I. BACKGROUD AND INTRODUCTION 1 1.1 An Inttoduction to Mammalian Fertilization 1 1.2 Species Specificity of Sperm-Egg Adhesion 4 1.3 Role of Zona Pellucida Glycoproteins in Sperm-Egg Adhesion 10 1.4 Controversial Data on Zona Pellucida-Binding Candidates in Spermatozoa 15 1.5 Preliminary Studies on Zonadhesin 20 1.6 Multimerization and Functions of VWD Domains in vWF and Mucins 25 1.7 Purpose of Study 29 II. EXPRESSION OF ZONADHESIN FRAGMENTS IN CULTURED MAMMALIAN CELLS 35 2.1 Introduction 35 2.2 Materials and Methods 37 2.2.1 Constmction of pCMV-5/DO-Dl Plasmid 37 iv 2.2.2 Transient Expression of rDO-Dl Zonadhesin in COS-1 Cells 38 2.2.3 Purification of rDO-Dl Zonadhesin from COS-1 Cell Extract 39 2.2.4 Purification of rDO-Dl Zonadhesin from Culture Media 40 2.2.5 Constmction of Recombinant Protein Containing Zonadhesin Mucin Domain 40 2.2.6 Constmction of Recombinant Protein Containing von Willebrand Factor Signal Peptide 41 2.2.7 Expression and Purification of the DO-Dl Fusion Protein 42 2.2.8 Preparafion of DO-Dl Antisera 42 2.2.9 Electrophoresis and Western Blotting 43 2.3 Results and Discussion 43 2.3.1 rDO-Dl Was Constmcted and Expressed in COS-1 Cultured Mammalian Cells 43 2.3.2 rDO-Dl Was Purified from Cell Extract and Culture Media by Ni^"^ Affinity Chromatography 45 2.3.3 Constmcts with Mucin Domain and Constmcts with von Willebrand Factor Signal Peptide Were Generated 46 III. HETEROGENEOUS PROCESSING AND ZONA PELLUCIDA- BINDING ACTIVITY OF PIG ZONADHESIN 55 3.1 Introduction 55 3.2 Materials and Methods 57 3.2.1 Isolafion of Sperm Membrane Fraction 57 3.2.2 Isolation of Zona Pellucida 58 3.2.3 Zona Pellucida-Binding Assays 59 3.2.4 Expression and Purification of DO-Dl Fusion Protein 60 3.2.5 Preparation of DO-Dl Antisera 61 3.2.6 Expression and Purification of Dl and D3 Fusion Proteins 61 3.2.7 Preparation of Domain-Specific Antisera 62 3.2.8 Preparation of GST, GST-Dl, and GST-D3 Affinity Columns 62 3.2.9 Affinity Purification of Dl and D3 Antibodies 63 3.2.10 Preparation of D3 Immunoaffmity Column 63 3.2.11 Immunoaffmity Purification of Zonadhesin from Spermatozoa 64 3.2.12 Preparation of Antisera to Zonadhesin Holoprotein 65 3.2.13 Site-Directed Mutagenesis 65 3.2.14 Electrophoresis and Western Blotting 66 3.2.15 In FzYro Multimerization 67 3.2.16 Indirect Immunofluorescence 68 3.3 Results and Discussion 68 3.3.1 Zonadhesin Binding Sites Are Evenly Distributed on the Entire Zona Pellucida 68 3.3.2 Antibodies Were Developed to Characterize Zonadhesin Iso forms 69 3.3.3 Multiple Zonadhesin Forms Are Present in Freshly Ejaculated Spermatozoa 72 VI 3.3.4 The pi 05/45 Monomeric Form of Zonadhesin Binds Preferentially to the Zona Pellucida 74 3.3.5 Cysteines Other than Those in the CG(L/V)CG Motif Can Mediate Spontaneous Multimerization of Purified Zonadhesin Fragments/« Vitro 75 3.3.6. Zonadhesin Localizes to the Apical Heads of Pig Spermatozoa 77 3.3.7 Summary and Discussion 77 rv. TEMPORAL AND FUNCTIONAL CHARACTERIZATION OF ZONADHESIN PROCESSING AND LOCALIZATION 89 4.1 Introduction 89 4.2 Materials and Methods 93 4.2.1 Development of Anti-N Antisemm 93 4.2.2 Deglycosylation 94 4.2.3 Collection of Spermatozoa from the Epididymis 95 4.2.4 Detergent Extraction of Spermatozoa and Testis Tissue 95 4.2.5 Zona Pellucida Binding Assay 96 4.2.6 Immunoprecipitation 97 4.2.7 Electrophoresis and Westem Blotting 98 4.2.8 Bead Adhesion Assay 98 4.2.9 Indirect Immunofluorescence 99 4.2.10 Immunoelectron Microscopy 99 4.3 Results and Discussion 101 Vll 4.3.1 p300 Is a Processed Zonadhesin Gene Product That Includes a MAM Domain 101 4.3.2 p300 Includes a Mucin-Like Domain That is Glycosylated with 0-Linked Oligosaccharides 103 4.3.3 Both p45 and pl05 Are Glycosylated with N-linked Oligosaccharides 105 4.3.4 Zonadhesin Is Present in at Least Two Physicochemically Distinct Compartments of Pig Spermatozoa 106 4.3.5 p300 Undergoes Significant Changes during Sperm Maturation in the Epididymis, but p45 and p 105 Do Not 107 4.3.6 Oligomeric Changes of Zonadhesin Occur in the Epididymis 110 4.3.7 The Zona Pellucida-Binding Activity of Zonadhesin Increases in the Epididymis 112 4.3.8 Zonadhesin Localizes to the Outer Acrosomal Membrane and the Acrosomal Matrix by Immunoelectron Microscopy 113 4.3.9. Binding of Zonadhesin Is Sufficient to Support Adhesion of Cell-Sized Stmctures to the Zona Pellucida 115 4.3.10 Zonadhesin Is Processed by Hydrolysis of an Asp-Pro Bond 116 V. MARKED VARIATION IN THE BIOCHEMICAL PROPERTIES OF ZONADHESIN IN SPERMATOZOA FROM A VARIETY OF MAMMALS 135 5.1 Introduction 135 5.2 Materials and Methods 139 5.2.1 Collection of Spermatozoa 139 5.2.2 Differential Detergent Extraction of Spermatozoa 139 Vlll 5.2.3 Indirect Immunofluorescence 140 5.2.4 Immunoelectron Microscopy 140 5.3 Results and Discussion 141 5.3.1 Zonadhesin Proteins Vary Significantly among MammaHan Species 141 5.3.2 Zonadhesin Localizes to the Apical Head of Spermatozoa of All Tested Species 144 VI. SUMMARY AND DISCUSSION 152 6.1 Overview 152 6.2 How does Zonadhesin "See" the Zona Pellucida? 153 6.3 What Factor(s) Determine Zonadhesin's Species Specificity? 155 6.4 Future Studies 157 REFERENCES 160 IX ABSTRACT Fertilization is an important and unique process in organisms that generate offspring by sexual reproduction.