THE ACTIONS OF FIBROBLAST GROWTH FACTORS DURING PERI- IMPLANTATION CONCEPTUS DEVELOPMENT IN CATTLE By QIEN YANG 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 2010 1 © 2010 Qien Yang 2 To my parents, sister and wife 3 ACKNOWLEDGMENTS This dissertation would not have been possible without the expert guidance of my advisor Dr. Alan Ealy. I remember how great I felt when he accepted me as a PhD student in this lab even I knew nothing about molecular regulation of Interferon tau. He always has great patience, encourages me to try different ideas when there is a chance of failure, he always cares about his students and does his best to help us succeed. His guidance has been an invaluable experience and will be greatly appreciated throughout my scientific career. I still feel great to be his student and work in this fun lab. I would like to express my deep appreciation to all my committee members for their willingness to serve on the committee. Dr. Sally Johnson has been my second advisor and treated me as her own student. Her invaluable input and direction throughout my graduate career set the standard for me. Dr. Peter Hansen has allowed me to use his lab and learn embryo techniques; one chapter of this dissertation would not have been finished without the IVF system in his lab. Dr. Naohiro Terada’s expertise in embryonic stem cell biology has been an excellent source for me in the last 4 and half years. I would like to gratefully acknowledge our former technician Idania Alvarez. She taught me most of my lab skills, how to be a productive bench worker and how to be a good lab mate. Her help and encouragement will be appreciated forever. Special thanks also go to all the girl scientists in the Ealy lab. Flavia Cooke helped me with lab protocols when I first got here. Kathleen Pennington helped my English and converted me to be a football fan. Her friendship will be treasured throughout my lifetime. Thanks to Susan Rodgers, Teresa Rodina, Krista DeRespino, Jessica Van Scyoc, Claudia Klein and others for good discussion during the lab meetings and other occasions. They all 4 helped me improve my English and speaking skills. Mariana Giassetti did a preliminary study with me and conducted CT1 cell migration experiments. I owe my deepest gratitude to all current members in the Ealy and Hansen laboratories. Sarah Fields helped me on IVF and other experiments. One of the experiments described here would not be possible without her help. Thanks to Kun Zhang, we fortunately reunited at UF and worked together again. His kind help on many things will be appreciated. Dr. Manabu Ozawa’s knowledge and willingness to assist have pushed my research further. Special thanks to Manabu for his friendship, advice and support. Silvia Carambula always likes to share her research with us and lead very informative discussions. It is truly a pleasure to work with her. Paula Morelli always smiles and makes our lab a happy place. I also thank IVF master Luciano Bonilla for his generous help. I would regret of my doctoral years at UF if I did not join the Animal Molecular and Cellular Biology Graduate Program. Thanks all AMCB coordinators and conference committee for their hard work and excellent organization skills. I thank Dr. Peter Hansen, Dr. Badinga Lokenga and Ms. Joann Fischer. Life would be lonely without many friends I have made here at the University of Florida. Thanks to Dr. Changhao Bi, Ju Li, Kesi Liu, Sha Tao, Lilian Oliveira, Fei Chen, Haiyan Xing, Guizhi Zhu, Rong Shi, and John Midolo for their friendship and support. Finally, I would like to thank my parents, sister and wife. Words cannot express my gratitude and love to them. They are the reason I work hard and do what I should do every day. They are the reason I try my best to be a better researcher, a better person. 5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF FIGURES ........................................................................................................ 11 LIST OF ABBREVIATIONS ........................................................................................... 13 ABSTRACT ................................................................................................................... 15 CHAPTER 1 INTRODUCTION .................................................................................................... 17 2 LITERATURE REVIEW .......................................................................................... 21 Pre- and Peri-implantation Development ................................................................ 21 Conceptus Elongation in Ruminant Species..................................................... 21 Preimplantation development and genomic activation ............................... 22 Cell differentiation and blastocyst formation............................................... 23 Conceptus elongation and germ layer formation ........................................ 25 Embryo Implantation and Early Placentation .................................................... 26 Blastocyst activation for implantation in mice ............................................. 27 Embryo implantation in ruminant species .................................................. 28 The major types of placentation in different species .................................. 29 Molecular Regulation of Early Lineage Specification .............................................. 30 Specification of Trophoblast Cell Lineage ........................................................ 31 Asymmetric cell division and cell polarity ................................................... 31 Molecular control of trophectoderm specification ....................................... 31 Trophoblast cell differentiation ................................................................... 33 Molecular Regulation of Primitive Endoderm Development.............................. 35 Pluripotent stem cell self-renewal .............................................................. 35 Primitive endoderm specification ............................................................... 36 Primitive endoderm development in ruminant ............................................ 38 Control of Conceptus Development and Interferon tau Expression ........................ 40 Maternal Recognition of Pregnancy in Ruminants ............................................ 40 The function of interferon tau during early pregnancy ................................ 41 Transcriptional regulation of interferon tau production ............................... 43 Signaling pathways controlling interferon tau production ........................... 45 Regulation of Pre- and Peri-development by Growth Factors .......................... 46 Factors shown beneficial effects on preimplantation development ............ 47 Regulation of trophoblast cell migration ..................................................... 48 Uterine derived factors stimulate IFNT production ..................................... 50 Roles for FGFs in mediating conceptus development in ruminants ........... 51 6 3 PROTEIN KINASE C DELTA MEDIATES FIBROLBAST GROWTH FACTOR-2- INDUCED INTERFERON TAU EXPRESSION IN BOVINE TROPHOBLAST ........ 55 Materials and Methods ............................................................................................ 57 Materials ........................................................................................................... 57 Trophoblast Cell Culture ................................................................................... 58 Quantitative Real Time RT-PCR ...................................................................... 60 Western Blot Analyses ..................................................................................... 60 Antiviral Activity ................................................................................................ 62 Bovine Conceptus Collection and End-point RT-PCR ...................................... 63 RNA-interference .............................................................................................. 64 Statistical Analyses .......................................................................................... 64 Results .................................................................................................................... 65 ERK1/2- and p38 MAPK-dependent Regulation of IFNT Expression ............... 65 PKC-dependent Regulation of IFNT Expression .............................................. 66 PKC-delta Expression and Activation in Trophectoderm .................................. 68 Impact of PKC-delta Knockdown on Vivot Cell Responsiveness to FGF2 ....... 68 PKC-delta Regulates IFNT Expression through ERK1/2-independent Pathways ....................................................................................................... 69 Discussion .............................................................................................................. 70 4 PRIMITIVE ENDODERM DEVELOPMENT IS STIMULATED BY FIBROBLAST GROWTH FACTOR 2 IN BOVINE BLASTOCYSTS ............................................... 86 Materials and Methods ............................................................................................ 89 Materials ..........................................................................................................