Protein Contributions of the Male Accessory Organs to The
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Old Dominion University ODU Digital Commons Theses and Dissertations in Biomedical Sciences College of Sciences Winter 1983 Protein Contributions of the Male Accessory Organs to the Composition of Human Seminal Plasma as Determined by High Resolution Two- Dimensional Electrophoresis Edward E. Gaunt Old Dominion University Follow this and additional works at: https://digitalcommons.odu.edu/biomedicalsciences_etds Part of the Biology Commons, and the Surgery Commons Recommended Citation Gaunt, Edward E.. "Protein Contributions of the Male Accessory Organs to the Composition of Human Seminal Plasma as Determined by High Resolution Two-Dimensional Electrophoresis" (1983). Doctor of Philosophy (PhD), dissertation, Biological Sciences, Old Dominion University, DOI: 10.25777/bcd9-x288 https://digitalcommons.odu.edu/biomedicalsciences_etds/103 This Dissertation is brought to you for free and open access by the College of Sciences at ODU Digital Commons. It has been accepted for inclusion in Theses and Dissertations in Biomedical Sciences by an authorized administrator of ODU Digital Commons. For more information, please contact [email protected]. PROTEIN CONTRIBUTIONS OF THE MALE ACCESSORY ORGANS TO THE COMPOSITION OF HUMAN SEMINAL PLASMA AS DETERMINED BY HIGH RESOLUTION TWO-DIMENSIONAL ELECTROPHORESIS by Edward E. Gaunt B . S . , June, 1972 Virginia Polytechnic Institute and State University M.S., August, 1980 Old Dominion University A Dissertation Submitted to the Faculties of Eastern Virginia Medical School and Old Dominion University in Partial Fulfillment of the Requirements for the Degree of DOCTOR. OF I I. OSOFH Y BIOMEDICAL SCIENCES EASTERN VIRGINIA MEDICAL SCHOOL and OLD DOMINION UNIVERSITY December, 1983 Approved by: . vfc-f''Cha i rman 'Anj. Steven B. Ackerman, Ph.D. PIeban, Ph.D . J o h Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (£) Copyright by Edward E. Gaunt, 1983 . All rights reserved. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABSTRACT PROTEIN CONTRIBUTIONS OF THE MALE ACCESSORY ORGANS TO THE COMPOSITION OF HUMAN SEMINAL PLASMA AS DETERMINED BY HIGH RESOLUTION TWO-DIMENSIONAL ELECTROPHORESIS Edward E. Gaunt Old Dominion University and Eastern Virginia Medical School, December, 1983 Director: Dr. James H. Yuan Human Seminal Plasma was evaluated using a High Resolution Two-Dimensional Electrophoresis technique adapted by this laboratory. Seminal plasma from healthy volunteers with recently proven fertility were characterized using this technique to establish "normal" protein distribution patterns in the two-dimensional polyacrylamide gel slab. In addition, time studies were performed on selected specimens to determine the effects of liquefaction on seminal plasma protein composition within the first several hours after collection. Split ejaculates were collected for identification and elaboration of accessory organ components in the whole seminal plasma specimen. A more direct approach to accessory organ component analysis involved electrophoresis of semen from patients with known fertility disorders (i.e. reproductive organ dysgenesis or dysfunction), patients who have undergone surgical procedures (varicocele repair, vasectomy, trans-urethra 1 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. resection etc.), or specimens from patients who have produced secretions as a result of prostatic or seminal vesicle massage in the course of urological examinations. From these results, patterns of protein "spots" in the two-dimensional gel were associated with a particular accessory organ. Finally, seminal plasma from infertile individuals suffering from (idiopathic) polyzoospermia, hyperspermia, oligozoospermia, or azoospermia were evaluated. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DEDICATION I would like to dedicate this work to my wife Carol Marie as well as to three other individuals who have had a great influence on my academic career: Mr. Robert L. Householder who started me on this journey, Dr. John R. Vercellotti who gave me guidance along the way, and D r . James H . Yuan my advisor for the past six years. 1 1 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGEMENTS It's difficult to recognize ell of the individuals who have touched upon my life during the course of this study. The most important, of course, have been my wife Carol and my sons Kevin and Ryan. It is impossible for me to thank them for all of the many sacrifices they have made to get me "educated". Thanking them for their love, support, and constant encouragement doesn't even come close to expressing my gratitude and love for them. Dr. James Yuan has been my instructor, advisor, director, and councelor as well as a friend for the past six years. I'd like to thank him for the privilege of being his student, and for all of the support he has given me in the course of my studies and research. I'd also like to thank my Dissertation Guidance Committee members for the hours they've spent directing this research: Dr. Fat Fleban for her valuable advice, constant encouragement, and trace metal studies; Dr. Jack Stecker for providing a majority of the patient specimens and clinical histories as well as for the many discussions we had and the ideas he provided; and Dr. Steve Ackerman and Dr. Anibal Acosta for all of the patient and donor specimens they provided through the Andrology Lab and the Oligospermic Program, as well as for their guidance and direction. i i i Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. di rec t ion. Others who have contributed to this effort include Sheri Coble from the Urology Associates' laboratory, Kathy Flagge and Rosita Acosta from the ODU Andrology Lab, and the many "nameless" patients and donors who provided specimens for this research. The Old Dominion University Science Shop has provided excellent technical support in their construction of a majority of the electrophoresis chambers used in this study. Mr. John Hill is another individual who has provided outstanding technical support for this project. I'd like to thank him for both his invaluable electronic support, and his friendship. I'd also like to thank the Elders, Deacons, and members of the congregation of Kempsville Presbyterian Church for their prayers, love, and support. There have been many times when they have provided both spiritual and physical uplifting when "everything" appeared to be going wrong. A final note of thanks goes to my fellow graduate students - especially Steve Knizner, Hsing Ying [Jean] Liu, C.S. Cheung and Ying Lin. I'd like to thank them all for their camaraderie, encouragement, and suggestions, and for their help in monitoring experiments when I wasn't around. i v Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS Pa ge LIST OF F I G U R E S ...............................................vi i i LIST OF T A B L E S ............................................. iK Chap ter I. INTRODUCTION . 1 A. The Male Reproductive S y s t e m .......... 2 1. Developmental Aspects ............... 2 2. The Male Sen Organs and Semen Production .................... 4 3. The Production of S e m e n ............ 11 4. Semen Biochemistry ................. 14 B. Semen Analysis ............................ 21 1. Historical Aspects ................. 22 2. Traditional Semen Analysis .... 23 3. Biochemical Analysis ............... 24 4. High Resolution Two-Dimensional Electrophoresis ...................... 25 II. STATEMENT OF PURPOSE .............................. 2 9 III. EXPERIMENTAL TECHNIQUES ......................... 31 A . Ma te r i a l s ................................... 31 B. Equipment ................................... 31 C. Human Subjects ............................ 32 D. M e t h o d s ...................................... 33 1. Sample Collection .................... 33 2. Semen Analysis ...................... 35 v Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3. Seminal Plasma Denaturation .... 36 4. Isoelectric Focusing (IEF) .... 36 5. IEF Gel Equilibration............ 37 6. pH P r o f i l e ......................... 38 7. The Second Dimension SDS Slab Gel 38 8. Preparation for the Second Dimension Separation ............... 39 9. Slab Gel E l e c t r o p h o r e s i s ....... 39 10. Protein Visualization ............... 39 11. Protein Pattern Interpretation . 40 12. Photography......................... 40 IV. RESULTS ............................................. 42 A. Time Course S t u d i e s ....................... 42 B. Split Ejajulate Studies .................. 45 C. Other Seminal Fractions .................. 52 D. "Normal" Volunteer Studies............... 55 E. Patient S t u d i e s ............................ 57 1. Azoospermic Patients ............... 57 2. Post Vasectomy P a t i e n t s ......... 59 3. Varicocele Patients .................. 61 4. Other P a t i e n t s .................... 63 V. DISCUSSION ........................................... 67 VI . S U M M A R Y ............................................. 80 REFERENCES ................................................. 83 APPENDICIES A. Vertical Slab Gel Electrophoresis Chamber Specifications ..........................