University of New Hampshire University of New Hampshire Scholars' Repository Doctoral Dissertations Student Scholarship Fall 2007 Glycoprotein hormone receptors in sea lamprey (Petromyzon marinus): Identification, characterization and implications for the evolution of the vertebrate hypothalamic-pituitary-gonadal/thyroid axes Mihael Freamat University of New Hampshire, Durham Follow this and additional works at: https://scholars.unh.edu/dissertation Recommended Citation Freamat, Mihael, "Glycoprotein hormone receptors in sea lamprey (Petromyzon marinus): Identification, characterization and implications for the evolution of the vertebrate hypothalamic-pituitary-gonadal/ thyroid axes" (2007). Doctoral Dissertations. 395. https://scholars.unh.edu/dissertation/395 This Dissertation is brought to you for free and open access by the Student Scholarship at University of New Hampshire Scholars' Repository. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of University of New Hampshire Scholars' Repository. For more information, please contact [email protected]. GLYCOPROTEIN HORMONE RECEPTORS IN SEA LAMPREY (PETROMYZON MARI Nil S): IDENTIFICATION, CHARACTERIZATION AND IMPLICATIONS FOR THE EVOLUTION OF THE VERTEBRATE HYPOTHALAMIC-PITUITARY-GONADAL/THYROID AXES BY MIHAEL FREAMAT B.S. University of Bucharest, 1995 DISSERTATION Submitted to the University of New Hampshire in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy In Biochemistry September, 2007 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UMI Number: 3277139 INFORMATION TO USERS The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed-through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. UMI UMI Microform 3277139 Copyright 2007 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. This dissertation has been examined and approved. Dissertation Director, Dr. Stacia A. Sower Interim Associate Dean of Research of the College of Life Sciences and Agriculture, Professor of Biochemistry and Molecular Biology £ ,LQa Dr. William R^'frumble, Professor of Biochemistry and Molecular Biology ^ , / W > _____________ Dr. Clyde LMDenis, Professor of Biochemistry and Molecular Biology Dr. William A. Condon, Professor of Animal and Nutritional Sciences ^ ' A c -------------------- Dr. William R Moyle, University of Medicine and Dentistry of New Jersey, Professor of OBGYN c o } ~ Date Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGEMENTS I would like to thank: My adviser, Dr. Stacia A Sower, for giving me the possibility to work in her laboratory, for her continuous support in the lab, for her suggestions and advice in the scientific field and in the everyday life. Members of my Doctoral Committee: Dr. William Condon, Dr. Clyde Denis, Dr. William Trumble, Dr. William Moyle, Dr. Thomas Kocher for their help and understanding during my PhD stages. I would like to express my gratitude to Dr. William Moyle for his generous help with reagents and protocols, for the insightful discussions we had and for the thorough comments he made on my dissertation and research in general. All members of the Sower Lab, in particular Scott Kavanaugh for being the greatest colleague I ever worked with. Geoff Bushold, Matt Silver, Kazu Okamoto, Taka Kosugi, and many others, graduate and undergraduate students with whom I have shared the lab space, the reagents, the protocols, the ups and downs of a graduate student's life. My wife Dana for more than I probably realize. iii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS ACKNOWLEDGEMENTS..............................................................................................................iii LIST OF TABLES........................................................................................................................... vi LIST OF FIGURES........................................................................................................................vii ABSTRACT....................................................................................................................................ix CHAPTER PAGE INTRODUCTION............................................................................................................................1 The Vertebrate Hypothalamic-Pituitary-Gonadal (HPG) and Hypothalamic-Pituitary- Thyroid (HPT) Endocrine A xes ...............................................................................................2 Glycoprotein Hormone Receptors (GpH-R) Structure and Function .................................. 4 Evolution of Glycoprotein Hormone Receptors and Hypothalamic-Pituitary- Gonadal/Thyroid Axes ............................................................................................................11 The Sea Lamprey Petromyzon marinus and its Evolutionary Significance ......................16 Specific A im s ......................................................................................................................... 23 I. IDENTIFICATION AND CHARACTERIZATION OF THE LAMPREY GLYCOPROTEIN HORMONE RECEPTOR 1.......................................................................................................... 24 Introduction ............................................................................................................................ 24 Materials and Methods ......................................................................................................... 26 Results ....................................................................................................................................34 Discussion ............................................................................................................................. 38 II. IDENTIFICATION AND CHARACTERIZATION OF THE LAMPREY GLYCOPROTEIN HORMONE RECEPTOR II......................................................................................................... 45 Introduction ......................................................................................................................45 iv Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Materials and Methods .......................................................................................................... 47 Results ....................................................................................................................................57 Discussion .............................................................................................................................. 65 III. FUNCTIONAL DIVERGENCE OF GLYCOPROTEIN HORMONE RECEPTORS.........69 Introduction ............................................................................................................................ 69 Materials and Methods .......................................................................................................... 73 Results ....................................................................................................................................81 Discussion ...............................................................................................................................89 SUMMARY AND CONCLUSIONS..............................................................................................96 BIBLIOGRAPHY........................................................................................................................... 97 APPENDICES ...................................................................................................................... 114 APPENDIX A. IACUC Approval Note ...................................................................................... 115 APPENDIX B. cAMP Assay Using the pCRE-SEAP Reporter System ................................. 116 APPENDIX C. Oligonucleotides Used in the Study of Lamprey GpH-R I and I I .................. 122 APPENDIX D. Chemical Communication in Vertebrates .........................................................123 v Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF TABLES Table 1. Vertebrate glycoprotein hormone receptors molecular cloning ............... 12 Table 2. Constitutive activity of IGpH-R 1 .............................................................. 38 Table 3. Vertebrate GpH-Rs: database records, domain boundaries and IGpH-RI and II similarities ...................................................................................................61 Table 4. Transfection reagent amounts for pCRE-SEAP/receptor co-transfection. 118 Table 5. Oligonucleotides used in