ROLE OF THYROID HORMONES IN THE DEVELOPMENT OF GONADAL SEX, EXTERNAL MORPHOLOGY AND INTESTINAL SYSTEM OF ZEBRAFISH (DANIO RERIO) by PRAKASH SHARMA, B.S., M.S. A Dissertation In BIOLOGY Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Approved Dr. Reynaldo Patiño Chair of Committee Dr. Gregory D. Mayer Dr. James Carr Dr. Lauren Gollahon Dr. Nathan Collie Dr. Richard Strauss Dominick Joseph Casadonte Dean of the Graduate School December, 2012 Copyright 2012, Prakash Sharma Texas Tech University, Prakash Sharma, December 2012 ACKNOWLEDGMENTS First, I am privileged to thank Dr. Reynaldo Patiño, my major advisor and mentor for his guidance and encouragement which have been of immense support. He has been there to help and extend his guidance, anywhere, anytime, even at wee hours despite his busy schedule. Through him, I have not only picked up skills and a sense of responsibility, but also a whole new level of dedication. It is definitely a lifetime opportunity to have worked with an efficient and resourceful scientist like him. I am honored to thank my Committee Members for their enthusiasm to scrutinize my research. Thanks to Drs. Gregory D. Mayer, James Carr, Lauren Gollahon, Nathan Collie, and Richard Strauss. Their suggestions, ideas and information on the analysis of my study were of great significance. I could not have asked for a better cooperating team. Also, a million thanks to Dr. Gregory D. Mayer for supporting and guiding me immensely throughout my research program and for allowing me the opportunity to work in his lab. Of course, I cannot miss out wonderful people like Song Tang and Kaylyn Germ for helping, teaching and assisting, every time I needed them. I cannot miss the name of Dr. Timothy B Grabowski for constantly being supportive and encouraging. Without each one of you, it would have been a herculean task to do all the lab work alone. I will cherish your friendship and help. You will be remembered always. The contribution of Dr. Mayer and Song Tang to the chapter III and Dr. Grabowski to chapter IV was significant therefore I am including them as co-author in the respective chapters. ii Texas Tech University, Prakash Sharma, December 2012 I also acknowledge the Department of Biological Sciences of Texas Tech University for the financial support through a Teaching/Research Assistantship during my PhD program. This study was facilitated by intramural funding from Texas Tech University and Texas Cooperative Fish and Wildlife Research Unit (TCFWRU). TCFWRU cooperators include U.S. Geological Survey, Texas Tech University, Texas Parks and Wildlife Department and U.S. Fish and Wildlife Service. Special thanks from the core of my heart to all my dear lab mates: Dr. Leticia Torres, Jamie Suski, Matt VanLandeghem, Matt Meyer, Benard Ogola and Seydou Toe. Especially to Leticia, who not only helped me with the formatting but always lent her sisterly support through the course of my Research. My heartfelt gratitude to my dear family and friends for their immeasurable love, support and encouragement. Thank you from the depth of my heart, to my parents and in-laws, for being by my side. Their love and support have always kept me going though we were million miles away from each other. I am utterly grateful to my wife, Namrata; needless to say, she has been as much a part of my work as anybody else and has shown incredible zeal to ensure the success of my endeavour. She gave me all the love and strength I needed to withstand the challenges through the better part of my research and kept our marriage strong from continents away. In spite of all the hurdles that a long-distance marriage can bring, her love and encouragement have paved a way for boundless happiness and success. Lastly, to those who I may have missed thanking individually, I thank you all and you are as much a part of the success. iii Texas Tech University, Prakash Sharma, December 2012 TABLE OF CONTENTS ACKNOWLEDGMENTS………………………………………………………..……ii ABSTRACT ........................................................................................................... viii LIST OF TABLES ................................................................................................. xiv LIST OF FIGURES ................................................................................................. xv CHAPTER I. INTRODUCTION ................................................................................................... 1 The thyroid endocrine system of teleost fishes ..................................................... 1 Gonadal sex determination, differentiation and development ............................... 3 Gonadal sex determination and differentiation: gene cascades ..................... 4 Morphology of gonadal sex differentiation .................................................. 7 Gametogenesis and pubertal development ................................................... 7 Regulation of gonadal sex differentiation and development by thyroid hormone ...................................................................................................... 9 Metamorphosis .................................................................................................. 11 Teleost metamorphosis .............................................................................. 11 Regulation of teleost metamorphosis by thyroid hormone .......................... 13 Teleost gastrointestinal and hepatic development ...................................... 14 Regulation of teleost gastrointestinal development and hepatic function by thyroid hormone ........................................................................................ 16 Objectives and significance of present study ...................................................... 17 iv Texas Tech University, Prakash Sharma, December 2012 References .......................................................................................................... 19 II. REGULATION OF GONADAL SEX RATIOS AND PUBERTAL DEVELOPMENT BY THE THYROID ENDOCRINE SYSTEM IN ZEBRAFISH (DANIO RERIO) ...................................................................................................... 36 Abstract ............................................................................................................. 36 Introduction ....................................................................................................... 37 Methodology ...................................................................................................... 40 Preparation of treatment solutions ............................................................. 40 General husbandry .................................................................................... 41 Experimental design .................................................................................. 43 Histological methods ................................................................................. 43 Data analysis ............................................................................................. 44 Results ............................................................................................................... 45 Survival and growth .................................................................................. 45 Thyroid follicle condition .......................................................................... 46 Gonadal sex ratios and pubertal development ............................................ 47 Discussion ......................................................................................................... 49 References ......................................................................................................... 56 III. EFFECTS OF GOITROGEN AND THYROID HORMONE ON SEX RATIOS AND GENE EXPRESSION PROFILES DURING GONADAL SEX DIFFERENTIATION OF ZEBRAFISH (DANIO RERIO) ....................................... 71 Abstract ............................................................................................................. 71 Introduction ....................................................................................................... 72 v Texas Tech University, Prakash Sharma, December 2012 Methodology ..................................................................................................... 75 Treatment solution .................................................................................... 75 General husbandry .................................................................................... 76 Experimental design .................................................................................. 77 RNA isolation ........................................................................................... 78 Quantitative RT-PCR ................................................................................ 78 Gonadal histology ..................................................................................... 79 Data Analyses ........................................................................................... 80 Results ................................................................................................................ 81 Gonadal sex ratios and development at 45 dpf .......................................... 81 Gene expression ........................................................................................ 82 Discussion ........................................................................................................