THE EVOLUTION OF ENDOCRINE EXTRAEMBRYONIC MEMBRANES; A COMPARATIVE STUDY OF STEROIDOGENESIS AND STEROID SIGNALING IN THE CHORIOALLANTOIC MEMBRANE OF OVIPAROUS AMNIOTES By LORI CRUZE ALBERGOTTI A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2011 1 © 2011 Lori Cruze Albergotti 2 Ken, I will be forever grateful for your love and support. 3 ACKNOWLEDGMENTS First, I would like to thank Lou Guillette for his generous financial, technical and emotional support. Lou has taught me that being a scientist is the opportunity to be a detective in pursuit of answers to an intriguing question, and this pursuit requires not only an analytical mind, but also a creative one. I thank Lou for giving me this opportunity and for his endless guidance and encouragement, and last but not least for his friendship. I could not have asked for a better graduate experience and I am convinced that this has everything to do with having Lou as an advisor and Bernie, Colette, Marty and Malcolm as committee members. I would like to thank Bernie for pushing me outside of my comfort zone, for encouraging me to think broadly about my research, and for his willingness to provide lab assistance. Thank you to Colette for lending statistical and experimental design expertise, for pushing me to think about the big picture of my research, and for always believing in me. I would like to thank Marty for expanding my understanding of evolution and development, for allowing me the amazing opportunity to teach with him, and for making his lab available to me. Thank you to Malcolm for his expertise in developmental signaling, for his constant enthusiasm, and for his willingness to the take time to hear how things are going and offer words of encouragement. I would also like to thank the Department of Biology for providing a wonderful academic home for the last five years. I would like to thank the Biology faculty in general for being excellent academic role models and in particular I would like to thank Rebecca Kimball, Ed Braun, Charlie Baer, Marta Wayne and Dave Evans for taking an interest in me, offering guidance and assistance, and for helping me become a better scientist. I would also like to thank Keith Choe for the generous use of his microscope and camera. 4 Thank you to my lab mates, colleagues and friends from whom I have learned so much. Thanks to Satomi Kohno and Brandon Moore for teaching me molecular biology, for providing technical advice, and for helping me hit the ground running. Thank you to Thea Edwards and Alison Roark for sharing my interest in the placenta and extraembryonic membranes and for the countless hours of conversation that helped to define my ideas about the evolution of these structures. Thank you to Heather Hamlin for her assistance with tissue culture and hormone techniques, for her boundless energy and amazing spirit. Thank you to Krista McCoy, Leslie Babonis, Kelly Hyndman, and Marda Jorgensen for providing histology, immunohistochemistry and microscopy training and advice. Thank you to Mike McCoy for providing statistical expertise and support and for pushing me to think outside the box about data analysis. Thank you to Ashley Boggs and Nicole Botteri for sharing this experience with me and for their continuous support and friendship. Thanks to the wonderful undergraduate students that I have had the pleasure to mentor and without whose toiling hours in the lab this work would have been incredibly difficult; Chris Olmo, Yao Fu, Sasha Strul, Jacob Fyda, Shellah Palmer, Jenna Harty, Momna Younas, Gabrielle Rolland, and Patpilai Kasinpila. I would also like to thank the Florida Fish and Wildlife Conservation Commission for their assistance in the collection of alligator and turtle eggs. Finally, I would like to thank my mom and dad for their love and encouragement. A special thanks to all of my friends, but especially Chip, Michelle, Jon, Christine, Stephanie and the EC, for keeping me sane these last few years. I thank my wonderful husband for making me laugh every day, for reminding me that life is a balance and for his willingness to join in this adventure with me. 5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 9 LIST OF FIGURES ........................................................................................................ 10 LIST OF ABBREVIATIONS ........................................................................................... 12 ABSTRACT ................................................................................................................... 16 CHAPTER 1 INTRODUCTION .................................................................................................... 19 The Evolution of Viviparity ...................................................................................... 20 Egg Retention and the Lengthening of Gestation ............................................. 21 The Reduction of the Eggshell ......................................................................... 24 The Emergence of the Placenta ....................................................................... 27 An Endocrine Placenta ........................................................................................... 30 A New Hypothesis for the Evolution of Endocrine Extraembryonic Membranes and the Placenta: Implications for the Evolution of Viviparity ............................... 32 Specific Hypotheses ............................................................................................... 33 Significance of Work ............................................................................................... 34 2 STEROIDOGENESIS AND STEROID SIGNALING IN THE CHORIOALLANTOIC MEMBRANE OF THE CHICKEN (GALLUS GALLUS) ........ 37 Materials and Methods............................................................................................ 39 CAM Collection ................................................................................................. 39 RNA Isolation and Reverse Transcription......................................................... 39 Real-time Quantitative Polymerase Chain Reaction (RT-qPCR) ...................... 39 Cloning and Sequencing of Plasmids ............................................................... 41 RT–qPCR Primers ............................................................................................ 41 Sexing of Embryos ........................................................................................... 41 In Vitro Explant Culture .................................................................................... 42 Immunohistochemistry and Microscopy............................................................ 42 Statistical Analysis ............................................................................................ 43 Results .................................................................................................................... 45 The Chicken CAM has the Required Molecular Mechanisms to Perform Steroidogenesis and Synthesis of Progesterone ........................................... 45 The Chicken CAM is Capable of In Vitro Progesterone Synthesis ................... 46 The Chicken CAM is Capable of Responding to P4 Signaling Through the Progesterone Receptor ................................................................................. 47 6 Discussion .............................................................................................................. 49 3 THE CHORIOALLANTOIC MEMBRANE OF THE AMERICAN ALLIGATOR (ALLIGATOR MISSISSIPPIENSIS) HAS THE CAPABILITY TO PERFORM STEROID BIOSYNTHESIS AND RESPOND TO STEROID HORMONE SIGNALING ............................................................................................................ 56 Materials and Methods............................................................................................ 60 Egg Collection and Sample Preparation ........................................................... 60 RNA Isolation and Reverse Transcription......................................................... 61 Real-time Quantitative Polymerase Chain Reaction (RT-qPCR) ...................... 61 RT-qPCR Normalization ................................................................................... 62 Cloning and Sequencing of Plasmids ............................................................... 63 Immunohistochemistry and Microscopy............................................................ 63 Statistical Analysis ............................................................................................ 64 Results .................................................................................................................... 66 Internal Control Expression among Embryonic Stages and between Incubation Temperatures .............................................................................. 66 Steroidogenic Factor, Enzyme and Steroid Receptor Expression among Embryonic Stages ......................................................................................... 67 Steroidogenic Factor, Enzyme and Steroid Receptor Expression between Incubation Temperatures .............................................................................