The$Effects$of$Gestational$and$Lactational$Bisphenol$A$Exposure$on$Rat$Pup$Morphometric$ Measurements$and$on$Adrenal$Gland$Glucocorticoid$Receptor$Gene$Expression$ $ $ $ $ $ Julia&Hajjar& $ $ & Supervisors:&Dr.&Anne&T.M.&Konkle&and&Dr.&Karen&P.&Phillips& $ $ $ $ $ $ $ $ $ $ $ $ $ School&of&Interdisciplinary&Health&Sciences& Faculty&of&Health&Sciences& University&of&Ottawa& May&2017& $ $ $ $ $ $ $ $ $ $ $ Submitted&to&the&Faculty&of&Graduate&and&Postdoctoral&Studies&in&partial&fulfillment&of&the& requirements&of&the&degree&of&Master&of&Science&in&Interdisciplinary&Health&Sciences& & & & ©&Julia&Hajjar,&Ottawa,&Canada,&2017 & $ Table of Contents Acknowledgements ................................................................................................................. v Abbreviations ........................................................................................................................... vi List of Figures ......................................................................................................................... vii List of Tables and Appendices ................................................................................................ viii Abstract ................................................................................................................................... ix Background ............................................................................................................................. x PART I: Introduction ........................................................................................................ 1 Hypothalamic-Pituitary-Adrenal (HPA) Axis ........................................................................ 1 Glucocorticoids ...................................................................................................................... 3 Glucocorticoid Receptor ......................................................................................................... 4 Negative Regulation of the HPA axis ..................................................................................... 7 Estrogen Modulation of the HPA axis .................................................................................... 9 Perinatal Development of the HPA axis…………………………………………………… 12 Fetal Stress Response During Pregnancy ............................................................................... 13 Role of Placenta on Fetal HPA axis Development ................................................................. 15 Mechanisms to Protect Fetus during Pregnancy and Perinatal Stress .................................... 18 CRH-Binding Protein .............................................................................................................. 18 11β-Hydroxysteroid Dehydrogenase ...................................................................................... 19 Maternal Care and Pup HPA axis Hyporesponsiveness ......................................................... 20 Stress Hyporesponsive Period (SHRP) ................................................................................... 21 Corticosteroid Binding Globulin Hypothesis .......................................................................... 22 Anterior Pituitary Hypothesis ................................................................................................. 23 Adrenal Cortex Hypothesis ..................................................................................................... 24 Human Stress Hyporesponsive Period .................................................................................... 26 Estrogenic Endocrine Disrupting Chemicals .......................................................................... 27 Xenoestrogens ......................................................................................................................... 28 Diethylstilbestrol ..................................................................................................................... 28 Bisphenol A ............................................................................................................................ 29 Bisphenol A Mechanism ......................................................................................................... 30 Bisphenol A Metabolism ........................................................................................................ 31 Canadian Exposure to Bisphenol A ........................................................................................ 33 Significance of Bisphenol A ................................................................................................... 34 Research Question ................................................................................................................. 35 PART II : Materials and Methods .................................................................................. 36 Strain Selection ....................................................................................................................... 36 Ethics ....................................................................................................................................... 36 Animal Husbandry .................................................................................................................. 37 Exposure Model .................................................................................................................... 37 Dosing ..................................................................................................................................... 37 Exposure Solutions ................................................................................................................. 38 ii& & Exposure Protocol ................................................................................................................... 39 Sacrifice .................................................................................................................................. 39 Morphometric Measurements ............................................................................................. 40 Dams ....................................................................................................................................... 40 Offspring ................................................................................................................................. 41 Litter Size, Sex Ratio, Bodyweights ....................................................................................... 41 Crown-Rump Length .............................................................................................................. 41 Anogenital Distance ................................................................................................................ 41 Data Analysis .......................................................................................................................... 42 Dam Bodyweight Data Analysis ............................................................................................. 42 Offspring ................................................................................................................................. 43 Litter Size Data Analysis ........................................................................................................ 43 Sex Distribution Data Analysis ............................................................................................... 43 Morphometric Data Analysis .................................................................................................. 43 Pup Bodyweight Data Analysis .............................................................................................. 44 Pup Bodyweight Progression Data Analysis .......................................................................... 44 Crown-Rump Length Data Analysis ....................................................................................... 45 Anogenital Distance Data Analysis ........................................................................................ 45 Molecular Characterization of Glucocorticoid Receptor .................................................. 46 Tissue Collection .................................................................................................................... 46 RNA Isolation ......................................................................................................................... 46 RNA Concentration and Quality ............................................................................................. 47 RNA Gel Electrophoresis ....................................................................................................... 47 Reverse Transcription ............................................................................................................. 48 Design of Target Gene Primers ............................................................................................... 48 Reference Gene Primer Design ............................................................................................... 49 Primer Validation .................................................................................................................... 51 Quantitative Polymerase Chain Reaction (qPCR) .................................................................. 52 qPCR Plate Setup ...................................................................................................................