Development and testing of gene expression biomarkers for gonadal dysgenesis in conjunction with the US EPA Endocrine Disruptor Screening Program’s Tier 2 Larval Amphibian Growth and Development Assay A Thesis SUBMITTED TO THE FACULTY OF UNIVERSITY OF MINNESOTA BY Jonathan T. Haselman IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE Dr. Sigmund J. Degitz, Dr. Patrick Schoff, Dr. Kendall Wallace April 2014 © Jonathan T. Haselman 2014 Acknowledgements I would like to thank Dr. Allen Olmstead for his mentorship and review of Chapter 1, Dr. Rodney Johnson for his mentorship and camaraderie through Tier 2 development, Chad Blanksma for providing histotechnical support and micrographs of Xenopus tropicalis ovary and testis tissues, Hongzu Ren for processing and imaging the microarrays, and Dr. Marshall Hampton for guidance with writing Python code. I would also like to thank Dr. Daniel Villeneuve for advice regarding microarray and thoughtful comments and suggestions to improve Chapter 1. Many thanks to Pat Kosian for analytical chemistry and moral support and to Joe Korte for advice regarding quantitative PCR and help with stress relief at the curling club. I would like to express my immense gratitude to Joe Tietge for supporting my academic, professional and personal endeavors. I would especially like to thank my advisor, mentor, colleague and friend Sig Degitz for cajoling me to reach my full potential and for being my greatest advocate. To the rest of my advisory committee, Dr. Ken Wallace and Dr. Pat Schoff, thank you for supporting my extended schedule and believing that I would someday finish the M.S. toxicology degree program. i Dedication This thesis is dedicated to my loving and supportive wife who is now carrying our first child... and to my parents, in-laws, siblings, family, friends, mentors, Earth – thank you all for being part of this life. ii Abstract The Endocrine Disrupter Screening Program of US EPA has recently developed a Tier 2 testing guideline using model amphibian species Xenopus laevis. The Larval Amphibian Growth and Development Assay assesses a chemical’s endocrine-related effects in vivo and generates concentration-response information for ecological risk-assessment. Currently, the assay relies on histopathological evaluations for identifying endocrine- related reproductive effects. However, histopathology can seldom define the chemical mode of action and is not easily interpreted in the context of risk assessment when the effects are minimal to moderate. This study explores the use of gene expression biomarkers in the gonad that could potentially inform a chemical’s mode of action and detect adverse reproductive effects that are otherwise uncharacterized by histopathology. To identify candidate biomarkers, global expression was analyzed in differentiating ovary and testis tissues of Xenopus tropicalis. Genetic programs responsible for reproductive maturation in gonad tissues were characterized and provided a foundation from which specific genes could be selected proximal to a model chemical’s known mode of action. Four genes were selected within the putative androgen molecular network to evaluate as biomarkers of the anti-androgen mode of action characteristic of a common fungicide, prochloraz. Following continuous exposure to prochloraz throughout embryonic, larval and juvenile development in Xenopus laevis, assessments of growth, liver and kidney pathology, and reproductive development were made. To evaluate the predictive capabilities of the candidate genes, one gonad was kept in situ for histopathological evaluations while the other was processed for mRNA analyses. Results indicate that prochloraz exposure caused metabolic toxicity in the liver and kidney; it caused testis degeneration coincident with the onset of androgen-mediated spermatogenesis and inhibited regression of Müllerian ducts. Two of the four candidate genes showed increases in expression at the high test concentration and appeared to be predictive of an anti-androgen-induced adaptive response. The behavior of these biomarkers stimulated valuable discussion and generated testable hypotheses to better understand the evolution of molecular mechanisms driving gonad development in a cross-species context. This study provides a model for expression-based biomarker development in endocrine tissues and offers direction toward enhanced ecological risk-assessment. iii Table of Contents Acknowledgements .............................................................................................................. i Dedication ........................................................................................................................... ii Abstract .............................................................................................................................. iii Table of Contents ............................................................................................................... iv List of Tables ..................................................................................................................... vi List of Figures ................................................................................................................... vii Overview ............................................................................................................................. 1 Background ..................................................................................................................... 1 Problem ........................................................................................................................... 2 Objectives ....................................................................................................................... 2 Approach ......................................................................................................................... 3 Chapter 1: Global gene expression analysis during early differentiation of Xenopus (Silurana) tropicalis gonad tissues. .................................................................................... 5 1. Introduction ................................................................................................................. 5 2. Materials and methods ................................................................................................ 8 2.1 Animal care and culture ........................................................................................ 8 2.2 Experimental design.............................................................................................. 9 2.3 Total RNA target preparation and hybridization .................................................. 9 2.4 Data analysis ....................................................................................................... 10 3. Results ....................................................................................................................... 12 3.1 Differential expression and clustering ................................................................ 12 3.2 Functional analyses ............................................................................................. 14 4. Discussion ................................................................................................................. 18 4.1 Ovary................................................................................................................... 19 4.2 Testis ................................................................................................................... 23 5. Conclusion ................................................................................................................ 27 Chapter 2. Measuring testis dysgenesis using gene expression biomarkers in Xenopus laevis following exposure to prochloraz. .......................................................................... 29 1. Introduction ............................................................................................................... 29 2. Materials and methods .............................................................................................. 33 2.1 Experimental design............................................................................................ 33 2.2 Test concentrations ............................................................................................. 33 2.3 Environmental conditions ................................................................................... 34 2.4 Exposure system ................................................................................................. 34 2.5 Chemical stock preparation ................................................................................. 35 2.6 Analytical chemistry ........................................................................................... 35 2.7 Test initiation ...................................................................................................... 36 2.8 Feeding ................................................................................................................ 37 2.9 Larval sub-sampling............................................................................................ 37 2.10 Cull .................................................................................................................... 38 2.11 Juvenile sampling (test termination) ................................................................. 38 2.12 Histological procedures ...................................................................................