MOLECULAR MECHANISM OF EMBRYO TRANSPORT AND EMBRYO IMPLANTATION IN MICE By SHUO XIAO (Under the Direction of Xiaoqin Ye) ABSTRACT Embryo transport and embryo implantation are essential events in mammalian reproduction during pregnancy. This dissertation was conducted to investigate the effect of bisphenol A (BPA) on early pregnancy and the molecular mechanism (s) of embryo transport and embryo implantation. Timed pregnant female mice were treated subcutaneously with 0, 0.025, 0.5, 10, 40, and 100 mg/kg/day BPA from gestation day 0.5 (D0.5, mating night as D0) to D3.5. High dose of preimplantation BPA exposure resulted in delayed embryo transport and preimplantation embryo development, and delayed/failed embryo implantation, indicating the adverse effect of BPA on early pregnancy. Preimplantation 17β-estradiol (E2) exposure at 1 and 10 µg/kg/day from D0.5 to D2.5 delayed embryo transport in the ampulla-isthmus junction of oviduct in mice, which is associated with the oviductal epithelium hyperplasia. Microarray analysis revealed 53 differentially expressed genes in the oviduct upon 10 µg/kg/day E2 treatment, which may have potential function in embryo transport regulation. Embryo implantation is a process that the receptive uterus accepts an embryo to implant into the uterine wall. Microarray analysis of the preimplantation D3.5 and postimplantation D4.5 uterine luminal epithelium (LE) identified 627 differentially expressed genes and 21 significantly changed signaling pathways upon embryo implantation. 12 of these genes were newly characterized and showed spatiotemporal expression patterns in the mouse periimplantation uterine LE. The most upregulated gene in the D4.5 LE Atp6v0d2 (34.7x) is a subunit of vacuolar-type H+-ATPase (V-ATPase) that regulates the cell acidification through ATP hydrolysis and proton translocation. LE acidification was significantly increased upon embryo implantation on D4.5 which was parallel with the differential expression pattern of Atp6v0d2. The V-ATPase inhibitor bafilomycin A1 inhibited embryo implantation and decreased LE acidification, indicating the critical role of LE acidification in embryo implantation. The most downregulated gene in the D4.5 LE N- acetylneuraminate pyruvate lyase (Npl) (35.4x) is highly expressed in D2.5 and D3.5 uterine LE, and was significantly decreased on D4.5. This spatiotemporal expression pattern of Npl is progesterone receptor mediated. However, Npl mutant females showed normal embryo implantation and fertility, indicating the dispensable role of Npl in female reproduction. INDEX WORDS: Embryo transport, embryo implantation, preimplantation embryo development, fertility, bisphenol A, oviduct, uterus, uterine luminal epithelium, uterine receptivity, microarray analysis, vacuolar-type H+-ATPase, LE acidification, fat pad injection, N-acetylneuraminate pyruvate lyase, progesterone receptor, MOLECULAR MECHANISM OF EMBRYO TRANSPORT AND EMBRYO IMPLANTATION IN MICE By SHUO XIAO B.S., Peking University, China, 肖硕 2006 M.S., Peking University, China, 肖硕 2008 A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY ATHENS, GEORGIA 2013 © 2013 Shuo Xiao All Rights Reserved MOLECULAR MECHANISM OF EMBRYO TRANSPORT AND EMBRYO IMPLANTATION IN MICE By SHUO XIAO Major Professor: Xiaoqin Ye Committee: Julie A Coffield Nick M. Filipov Mary Alice Smith Jia-sheng Wang Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia August 2013 DEDICATION To my family iv ACKNOWLEDGEMENTS During the past five years of my Ph.D. study, I have been supported by many people on my life, study, research. The following dissertation will not be possible without your help. First, I would like to express my sincere appreciation to my major advisor Dr. Xiaoqin Ye. Her continuous support and help make me realize how to do a good research and be a good scientist. Actually, she is not only an academic advisor for me, but also a very good friend in my life. I also want to thank my advisory committee members: Dr. Julie A Coffield, Dr. Nick M. Filipov, Dr. Mary Alice Smith and Dr. Jia- sheng Wang. Thank you all very much for your advice and help on my research and study. All my lab mates, I really had a wonderful time with you all in Dr. Ye’s lab during the past five years. A special gratitude to Honglu, who taught me a lot when we were working together in Dr. Ye’s lab. I appreciate your time and patience, and wish you have a very good career and life in China. Jennifer, I want to thank you very much for your time and advice on my dissertation, which improves my English writing skills a lot. At last but not least, I want to thank Joanne Mauro, Kali King, and Misty Patterson in Interdisciplinary Toxicology Program and Department of Physiology and Pharmacology, and all the people who help me directly or indirectly. This dissertation would not have been possible without your help! v TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ............................................................................................... v LIST OF TABLES ............................................................................................................ ix LIST OF FIGURES .......................................................................................................... x CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW .................................................... 1 1.1 Fertilization and preimplantation embryo development ............................... 1 1.2 Oviduct and embryo transport ..................................................................... 1 1.3 Embryo implantation and uterine receptivity ................................................ 5 1.4 Effect of bisphenol A (BPA) on the female reproductive system ............... 17 1.5 Hypothesis ................................................................................................. 22 2 PREIMPLANTATION EXPOSURE TO BISPHENOL A (BPA) AFFECTS EMBRYO TRANSPORT, PREIMPLANTATION EMBRYO DEVELOPMENT, AND UTERINE RECEPTIVITY IN MICE ...................................................................... 25 2.1 Abstract ..................................................................................................... 26 2.2 Introduction ................................................................................................ 26 2.3 Materials and Methods .............................................................................. 29 2.4 Results ...................................................................................................... 32 2.5 Discussion ................................................................................................. 42 3 MOLECULAR MECHANISM OF DELAYED EMBRYO TRANSPORT IN OVIDUCT IN MICE ................................................................................................................ 48 3.1 Abstract ..................................................................................................... 48 vi 3.2 Introduction ................................................................................................ 49 3.3 Materials and Methods .............................................................................. 51 3.4 Results ...................................................................................................... 53 3.5 Discussion ................................................................................................ 64 4 DIFFERENTIAL GENE EXPRESSION PROFILING OF MOUSE UTERINE LUMINAL EPITHELIUM DURING PERIIMPLANTATION .................................. 72 4.1 Abstract ..................................................................................................... 72 4.2 Introduction ................................................................................................ 73 4.3 Materials and Methods .............................................................................. 75 4.4 Results ...................................................................................................... 77 4.5 Discussion ................................................................................................ 88 5 ACIDIFICATION OF UTERINE LUMINAL EPITHELIUM IS CRITICAL FOR EMBRYO IMPLANTATION IN MICE ................................................................ 97 5.1 Abstract ..................................................................................................... 97 5.2 Introduction ................................................................................................ 99 5.4 Materials and Methods ............................................................................ 101 5.4 Results .................................................................................................... 107 5.5 Discussion .............................................................................................. 117 6 PROGESTERONE RECEPTOR-MEDIATED REGULATION OF N- ACETYLNEURAMINATE PYRUVATE LYASE (NPL) IN MOUSE UTERINE LUMINAL EPITHELIUM AND NONESSENTIAL ROLE OF NPL IN UTERINE FUNCTION ........................................................................................................ 123 6.1 Abstract ................................................................................................... 124 vii 6.2 Introduction .............................................................................................. 125 6.3 Materials and Methods ...........................................................................