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The Sertoli Cell-Spermatid Junctional Complex: a Potential Avenue for Male Contraception

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The Sertoli Cell-Spermatid Junctional Complex: a Potential Avenue for Male Contraception University of South Florida Scholar Commons Graduate Theses and Dissertations Graduate School 10-11-2006 The Sertoli Cell-Spermatid Junctional Complex: A Potential Avenue For Male Contraception Katja Margrit Wolski University of South Florida Follow this and additional works at: https://scholarcommons.usf.edu/etd Part of the American Studies Commons Scholar Commons Citation Wolski, Katja Margrit, "The Sertoli Cell-Spermatid Junctional Complex: A Potential Avenue For Male Contraception" (2006). Graduate Theses and Dissertations. https://scholarcommons.usf.edu/etd/2762 This Dissertation is brought to you for free and open access by the Graduate School at Scholar Commons. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Scholar Commons. For more information, please contact [email protected]. The Sertoli Cell-Spermatid Junctional Complex: A Potential Avenue For Male Contraception by Katja Margrit Wolski A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Pathology and Cell Biology College of Medicine University of South Florida Major Professor: Don F. Cameron, Ph.D. Stanley J. Nazian, Ph.D. Christopher Phelps, Ph.D. Marzenna Wiranowska, Ph.D. Date of Approval: October 11, 2006 Keywords: ectoplasmic specialization, cell-cell junction, cell culture, micropipette, Adjudin © Copyright 2006, Katja Margrit Wolski DISCLOSURE Results from some of the experiments required the use of color in the technique to analyze the data. If color cannot be seen in this version of the dissertation, the color copy can be found at the Shimberg Health Sciences Library, 12901 Bruce B. Downs Blvd., Tampa, Florida 33612-4479, (813) 974-2243. DEDICATION Wir haben soviel Ungeklärtes auf dieser Welt, und damit dieses so bleibt, haben wir die Wissenschaft. - Otto Waalkes The important thing in science is not so much to obtain new facts as to discover new ways of thinking about them. - William Lawrence Bragg ACKNOWLEDGEMENT S I would like to thank the following people, without whom this degree would not be: Dr. Don Cameron for accepting me into his laboratory and for all his dedication, help, and mentorship over the past 6 years; the Deutscher Akademischer Austauschdienst and Prof. Dr. Christiane Kirchhoff at the Universität Hamburg/Universitätsklinikum Hamburg-Eppendorf for giving me the opportunity to join her laboratory for 7 months, where I learned a lot about myself as a scientist and gained much maturity; my mother for instilling in me the desire to make myself better; Brian for pounding into my head that I am worthy of this and for being patient with me; my committee members at the University of South Florida – Dr. Stanley Nazian, Dr. Christopher Phelps, and Dr. Marzenna Wiranowska – for reviewing this dissertation and pushing me; Dr. Barry Hinton from the University of Virginia for serving as outside chair; Cecile Perrault at the University of Florida for helping me set up the micropipette; everyone I have worked with and who has helped me throughout my graduate years at the University of South Florida and the Universität Hamburg/Universitätsklinikum Hamburg-Eppendorf; all the friends and family who have supported and encouraged me in my pursuit of higher education; and finally, me for doing what I was told, but also being stubborn, and for not giving up, even though at times the future looked bleak. TABLE OF CONTENTS List of Tables .......................................................................................................................iv List of Figures.......................................................................................................................v Abstract............................................................................................................................... 1 Chapter 1 – Background ..................................................................................................... 3 Spermatogenesis .................................................................................................... 4 Hormonal Control of Spermatogenesis ...................................................... 5 Hormonal Disruption of Spermatogenesis.................................................. 7 Sertoli Cells ............................................................................................................. 7 Sertoli Cell Cytoskeleton............................................................................. 8 Blood-Testis Barrier .................................................................................. 10 Sertoli Cell-Germ Cell Junctions........................................................................... 11 Anchoring Junctions.................................................................................. 12 Adherens Junctions. ..................................................................... 12 Cadherin-Catenin Complex. ............................................. 12 Nectin-Afadin-Ponsin Complex......................................... 17 Integrin Complex ............................................................... 18 Ectoplasmic Specialization ............................................... 20 Espin ..................................................................... 22 Tubulobulbar Complexes.................................................. 23 Focal Contacts .............................................................................. 24 Desmosomes ................................................................................ 25 Hemidesmosomes ........................................................................ 25 Gap Communicating Junctions................................................................. 26 Male Infertility and Adjudin.................................................................................... 27 Specific Aims and General Hypothesis ................................................................ 31 References............................................................................................................ 33 Chapter 2 – Strength Measurement of the Sertoli-Spermatid Junctional Complex ....................................................................................................................... 62 Abstract................................................................................................................. 63 Introduction ........................................................................................................... 64 Materials and Methods.......................................................................................... 67 Sertoli Cell Isolation, Culture, and Pre-Treatment.................................... 67 Spermatocyte and Round Spermatid Isolation and Unit Gravity Velocity Sedimentation ........................................................................................... 67 Sertoli-Germ Cell Coculture...................................................................... 68 Measurement of Junctional Strength Using a Micropipette Pressure Transducing System (MPTS).................................................................... 68 i The Micropipette ....................................................................................... 69 The Water Reservoir System.................................................................... 70 Cell Measurements ................................................................................... 70 Statistics.................................................................................................... 71 Results .................................................................................................................. 72 Discussion............................................................................................................. 74 References............................................................................................................ 77 Chapter 3 – The Sertoli-Spermatid Junctional Complex Adhesion Strength Is Affected in vitro By Adjudin.......................................................................................... 82 Abstract................................................................................................................. 83 Introduction ........................................................................................................... 85 Materials and Methods.......................................................................................... 87 Sertoli Cell Isolation, Culture, and Pre-Treatment.................................... 87 Round Spermatid Isolation and Unit Gravity Velocity Sedimentation...... 88 Sertoli-Germ Cell Coculture...................................................................... 88 Addition of Adjudin to the Coculture ......................................................... 89 Measurement of Junctional Strength Using a Micropipette Pressure Transducing System (MPTS).................................................................... 89 Viability/Cytotoxicity Assays ..................................................................... 90 Results .................................................................................................................. 92 Discussion............................................................................................................. 95 References...........................................................................................................
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