OVERCOMING ENDOMETRIOSIS-ASSOCIATED PREIMPLANTATION EMBRYO DEVELOPMENTAL ANOMALIES BY CULTURE _______________________________________ A Thesis presented to the Faculty of the Graduate School at the University of Missouri-Columbia _______________________________________________________ In Partial Fulfillment of the Requirements for the Degree Master of Science _____________________________________________________ by JUSTIN MEREDITH HENNINGS Dr. Kathy L. Sharpe-Timms, Thesis Supervisor DECEMBER 2013 The undersigned, appointed by the dean of the Graduate School, have examined the thesis entitled OVERCOMING ENDOMETRIOSIS-ASSOCIATED PREIMPLANTATION EMBRYO DEVELOPMENTAL ANOMALIES BY CULTURE presented by Justin M. Hennings, a candidate for the degree of master of science, and hereby certify that, in their opinion, it is worthy of acceptance. Dr. Kathy L. Sharpe-Timms Dr. Peter Sutovsky Dr. Randall Prather ACKNOWLEDGEMENTS To begin with, I would like to thank my graduate thesis committee, composed of Drs. Kathy L. Sharpe-Timms, Peter Sutovsky, and Randall Prather, for all of the advice and guidance that they have given me over the last couple of years. Dr. Prather has helped me to understand the intricacies behind embryo culture media. Dr. Sutovsky has been kind enough to contribute his extensive knowledge of fluorescent microscopy and imaging techniques, aiding in my pursuit to photograph and analyze hundreds of embryos. I am also very appreciable for the trust that Dr. Sutovsky and his entire laboratory team have put in me by allowing my use of their equipment, including the Nikon 800 fluorescent scope. Also - thanks to Miriam Sutovsky for her patience in training me to fix and process my embryo samples, along with Dr. Young-Joo Yi, Wonhee Song, and TJ Miles for helping me in navigating their laboratory! Finally: I need to extend a heartfelt ‘thank you’ to my advisor, Dr. Kathy Timms. Two years ago, Dr. Timms gave me an amazing opportunity by taking me on as a graduate student in her laboratory. Not only has she trained me to become a better researcher, advising me on how to progress with experiments and verbally present my research, but also a better person. She was capable of increasing my confidence level as a scientist, leader, and a member of the community. I would honestly not be the person that I am today without her mentorship and friendship. ii I would also like to acknowledge Natalia Kesserova and Carol Okamura of the University of Missouri Transgenic Core for providing us with the culture media necessary for my experiments. Thank you to Randy Zimmer for manning the anesthesia machine during all of our experimental surgeries, and volunteering his expertise to help us collect embryos during the early hours of the morning! Thank you to Henda Nabli for all of the training, support, and assistance that she has given me over the last two years. I could not have gotten through a few of the ‘rougher patches’ of graduate school without her around. Thank you to all of the undergraduates who have cycled through our laboratory, helping with animal care, experimental surgeries, and tube labeling (Emily Rigden, Bailey Martin, Emma Windham, Hillary Moore, Lauren, Alexis Ruffolo, and Raven Wright). Finally – thank you to my parents (Larry and Marisa Hennings), aunt and uncle (Susan and Dale Wittmer), and brother (Travis Hennings) for all of the support and kind words that they have given me during my extensive time as a student. iii TABLE OF CONTENTS ACKNOWLEDGEMENTS ...................................................................................... ii LIST OF FIGURES .............................................................................................. vi LIST OF TABLES .............................................................................................. viii ABBREVIATIONS ................................................................................................ x CHAPTER ONE: A REVIEW OF THE LITERATURE .......................................... 1 ENDOMETRIOSIS ........................................................................................ 2 ENDOMETRIOSIS ETIOLOGICAL HYPOTHESES ...................................... 2 SYMPTOMS OF ENDOMETRIOSIS............................................................. 5 TREATMENTS OF ENDOMETRIOSIS ....................................................... 10 ANIMAL MODELS OF ENDOMETRIOSIS .................................................. 12 FERTILIZATION ........................................................................................ 13 OVIDUCTAL DYNAMICS ............................................................................ 15 EARLY STAGE PREIMPLANTATION DEVELOPMENT (ONE CELL TO MORULA) .......................................................................... 16 LATER STAGE PREIMPLANTATION DEVELOPMENT (MORULA TO BLASTOCYST) .................................................................... 18 METABOLISM AND PREIMPLANTATION DEVELOPMENT ..................... 21 IN VITRO AND CELL CULTURE MEDIA CONDITIONS ............................ 24 UBIQUITIN-PROTEASOME SYSTEM ........................................................ 33 PROJECT GOALS ...................................................................................... 45 CHAPTER TWO: PREIMPLANTATION EMBRYONIC DEVELOPMENT OF RAT ZYGOTES TRANSFERRED FROM AN IN VIVO ENDOMETRIOTIC ENVIRONMENT TO AN IN VITRO CULTURE SYSTEM ................................... 50 INTRODUCTION ........................................................................................ 51 MATERIALS AND METHODS .................................................................... 53 iv RESULTS ................................................................................................... 82 DISCUSSION ............................................................................................ 101 CHAPTER THREE: COMPARING THE QUALITY OF BLASTOCYST DEVELOPMENT IN A SEQUENTIAL CULTURE SYSTEM VERSUS A SINGLE CULTURE SYSTEM ........................................................................... 106 INTRODUCTION ....................................................................................... 107 MATERIALS AND METHODS .................................................................. 111 RESULTS ................................................................................................. 126 DISCUSSION ............................................................................................ 138 REFERENCES ................................................................................................. 143 VITA ................................................................................................................. 157 v LIST OF FIGURES Figure Page 1.1 Ubiquitin-Proteasome Schematic.................................................. 49 2.1 Experimental Groups .................................................................... 69 2.2 Embryo Culture System Schematic .............................................. 74 2.3 Timeline of Rat Embryo Culture ................................................... 75 2.4 Experimental Design ..................................................................... 76 2.5 Comparison of Embryo Fragmentation ......................................... 77 2.6 Stages of D2 Embryo Development .............................................. 78 2.7 Stages of D4 Embryo Development .............................................. 79 2.8 Nuclear Quality ............................................................................. 80 2.9 Other Embryonic Abnormalities .................................................... 81 2.10 Percentage of D2 Embryos with >50% Fragmentation ................. 84 2.11 Average Number of Nuclei Per D2 Embryo .................................. 86 2.12 Average Number of Abnormal Nuclei Per D2 Embryo .................. 88 2.13 Percentage of D4 Embryos with >50% Fragmentation ................. 92 2.14 D4 Average Number of Total Nuclei Per Embryo ......................... 94 2.15 D4 Average Number of Abnormal Nuclei Per Embryo .................. 96 2.16 D4 UCHL1 and UCHL3 Immunolocalization ................................ 98 2.17a Percentage of Embryos with Diffuse UCHL1 ................................ 99 2.17b Percentage of Embryos with Diffuse UCHL3 ................................ 99 3.1 Nuclear Morphology .................................................................... 123 3.2 Hatching vs. Unhatched Blastocysts ........................................... 124 3.3 Timeline of Murine Embryo Culture ............................................ 125 vi 3.4 Mean Nuclei Count Per Blastocyst ............................................. 128 3.5 Mean Number of TE and ICM Nuclei Per Blastocyst .................. 130 3.6 Mean Number of Abnormal and Normal Nuclei Per Blastocyst .. 132 3.7 Percentage of Hatched/Hatching Blastocysts ............................. 134 3.8 Percentage of Embryos at ≥8C (D3) or Blastocyst (D5) Stage ... 136 vii LIST OF TABLES Table Page 1.1 Precompaction Versus Postcompaction Embryo Requirements .................................................................................. 47 1.2 Media Component Comparison ....................................................... 48 2.1 Stages of Estrous ............................................................................ 68 2.2 Surgical Groups ............................................................................... 70 2.3 Rat Estrous Synchronization Schedul .............................................