GENE EXPRESSION PROFILING OF BOVINE OVARIAN FOLLICULAR SELECTION A Dissertation Presented to The Faculty of the Graduate School University of Missouri-Columbia In Partial Fulfillment Of the Requirement for the Degree Doctor of Philosophy By Zhilin Liu Dr. Eric Antoniou, Dissertation Supervisor December 2006 The undersigned, appointed by the Dean of the Graduate School, have examined the dissertation entitled Gene expression profiling of Bovine Ovarian Follicular Selection Presented by Zhilin Liu a candidate for the degree of Doctor of Philosophy and hereby certify that in their opinion it is worthy of acceptance. _______________________________________ Dr. Eric Antoniou _______________________________________ Dr. H. Allen Garverick _______________________________________ Dr. Gary S. Johnson _______________________________________ Dr. William R. Lamberson _______________________________________ Dr. Michael Smith _______________________________________ Dr. Jerry Taylor ACKNOWLEDGEMENTS First I would like to take this opportunity to express my appreciation to my advisor, Dr. Eric Antoniou, for letting me pursue my doctoral degree under his guidance. For more than six years, he has provided an encouraging environment for me to learn and grow in the research. His consistent guidance and cordial advice on my research have been invaluable. Many thanks to my committee members: Dr. Allen H. Garverick, Dr. William R. Lamberson, Dr. Gary S. Johnson, Dr. Michael Smith and Dr. Jerry Taylor for their encouragement and mentoring throughout my course of studies and completion of this work. Very special thanks to Dr. H. Allen Garverick and his lab member Malinda N. Burkhart, Dr. Robert S. Youngquist, Dr. Jerry Taylor and his lab member Erin M. Sellner, for their direct collaborations on different parts of this dissertation research. Without their generous help, this project would not be completed as smoothly as today. I would also like to thank Dr. Leonard B. Hearne for his expertise on microarray analysis and Dr. Dennis B. Lubahn for his concern and help. Many thanks to my current lab members, Sachin Bhusari, Mallikarjuna S. Basavarajappa, Jaewoo Kim as well as previous lab member, Jarrod J. Robertson for their help with science and lab work, and for creating an enjoyable lab together. I also need to extend my thanks to those who have been there along the way, including Raja S. Settivari, Dr. Kristi M. Cammack, Dr. Tim Safranski, Dr. Robert Schnabel, Jinghua Liu, Yi ii Zhuang and many other people in ASRC. Special thanks to Ms. Cynthia L. Glascock and Ms. Doris A. Lyons for their enormous assistance. Last, and certainly not least, appreciation is expressed to my loving and supporting family. I would like to express my deepest thanks to my parents and my sisters for their constant love and support throughout my life. Finally, and most importantly, none of this could have been completed without the love and support of my wife Wei Zhou. Thanks for all your help on science, for all the understanding and encouragement, for taking care of me and providing a warm home. You are the ones who make me a better person. Thanks. iii TABLE OF CONTENTS ACKNOWLEDGEMENTS........................................................................................... ii TABLE OF CONTENTS.............................................................................................. iv LIST OF TABLES.......................................................................................................viii LIST OF FIGURES....................................................................................................... ix LIST OF ABBREVIATION ......................................................................................... xi ABSTRACT .................................................................................................................xiii Chapter I ......................................................................................................................... 1 Introduction .................................................................................................................... 1 Chapter II........................................................................................................................ 4 Literature review............................................................................................................ 4 Introduction.................................................................................................................. 4 Follicular development ................................................................................................ 5 Preantral and antral follicle dynamics..................................................................... 5 Deviation and size advantage.................................................................................. 7 Gonadotropin regulation of follicular recruitment and selection............................ 8 Mechanisms of follicle selection ............................................................................... 12 FSH: follicle-coupling hypothesis ........................................................................ 12 LH and LH receptors ............................................................................................ 14 Estradiol ................................................................................................................ 15 Key steroidogenic enzymes .................................................................................. 18 TGFß superfamily members ................................................................................. 19 iv IGF-system............................................................................................................ 22 Role of macrophages............................................................................................. 27 Apoptosis .............................................................................................................. 29 Summary.................................................................................................................... 34 Chapter III .................................................................................................................... 36 Construction of a Bovine cDNA Array....................................................................... 36 Abstract...................................................................................................................... 36 Introduction................................................................................................................ 36 Results........................................................................................................................ 38 A. Construction of the array.................................................................................. 39 B. Sample labeling and hybridization................................................................... 45 Summary.................................................................................................................... 49 Chapter IV .................................................................................................................... 50 Data Storage and Bioinformatics Analysis................................................................. 50 Abstract...................................................................................................................... 50 Introduction................................................................................................................ 50 Results........................................................................................................................ 51 A. BASE for data storage and normalization........................................................ 51 B. MAANOVA for statistical analysis ................................................................. 52 C. TMEV for clustering ........................................................................................ 53 D. DAVID for gene list functional annotation...................................................... 53 E. Gene annotation................................................................................................ 54 Chapter V...................................................................................................................... 60 Molecular Mechanisms Regulating of Bovine Ovarian Follicular Selection .......... 60 Abstract...................................................................................................................... 60 v Introduction................................................................................................................ 61 Materials and methods............................................................................................... 63 Animals and treatments......................................................................................... 63 Tissue processing.................................................................................................. 64 Hormone analysis.................................................................................................. 64 RNA Extraction .................................................................................................... 65 Microarray preparation ......................................................................................... 65 Microarray hybridization .....................................................................................