INVESTIGATION OF FACTORS AFFECTING FERTILITY: CHROMOSOME SEGREGATION ERRORS AND ENVIRONMENTAL TOXINS by JODI M. JACKSON Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Thesis Advisor: Dr. Patricia A. Hunt Department of Genetics CASE WESTERN RESERVE UNIVERSITY August, 2007 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of ______________________________________________________ candidate for the Ph.D. degree *. (signed)_______________________________________________ (chair of the committee) ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. I dedicate this dissertation to the loving memory of my grandmother, Margaret Franz. Her unwavering conviction that education opens doors, coupled with her sacrifice to ensure her family had opportunities not available to her, propelled me far beyond where my own endurance surely would have failed. iv Table of Contents Dedication………………………………………………………………………………..iv Table of Contents………………………………………………………………………...v List of Tables……………………………………………………………………………..x List of Figures....................................................................................................................xi Abstract…………............................................................................................................xii Chapter 1: Introduction and Research Aims Introduction…………………………………………………………………..........1 Why Chromosome Segregation and Environmental Toxins?......................1 What Happens When Chromosome Segregation Goes Wrong?..................2 The BALB/cWt Y Chromosome………………………………………….3 Chromosome Segregation in Spo13 Deficient S. cerevisiae……………...4 What is a Centromere?.................................................................................5 Centromere Sequence…………………...………………...………6 Centromere Proteins………………………………………….……9 What is a Good Strategy to Study the Centomere?....................................13 What is Known About the Y Centromere?................................................13 Environmental Toxins and Human Reproduction……………………….16 Is the Incidence of Human Fertility Increasing?........................................16 Impacts of Environmental Toxins on Male Infertility…………………...17 Medical Exposure………………………………………………..17 Occupational Exposure…………………………………………..18 v Day-to-Day Exposure……………………………………………18 Impacts of Environmental Toxins on Female Infertility………………...22 Medical Exposure………………………………………………..22 Occupational Exposure…………………………………………..22 Day-to-Day Exposure……………………………………………23 What are Quaternary Ammonium Compounds?........................................24 Chapter 2: The mouse A/HeJ Y chromosome: Another good Y gone bad………..28 Abstract………………………………………………………………………….29 Introduction……………………………………………………………………...30 Methods and Materials…………………………………………………………..32 Mice…………………………………………………………….………..32 Southern Blot Analysis………………………………………….……….33 Fetal Gonad Analysis and Chromosome Preparations…………………..34 Fluorescent In Situ Hybridization Studies……………………………….35 Y Chromosome Mapping………………………………………..35 Aneuploidy Analysis…………………………………………….36 Meiotic Preparations…………………………………………………….36 Diakinesis and Metaphase II…………………………………….36 Pachytene………………………………………………………...37 Results…………………………………………………………………………...37 Hermaphroditism is a feature of the A/HeJ strain……………………….37 Hermaphroditism is a property of the A/HeJ Y chromosome…………...38 vi The YA/HeJ and YWt chromosomes are different on the sequence level….39 The YA/HeJ chromosome is prone to mitotic mis-segregation……………41 The YA/HeJ centromere exhibits morphological aberrations……………...42 The YA/HeJ chromosome segregates faithfully in meiosis………………..46 The structural integrity of the YA/HeJ chromosome………………………48 Discussion………………………………………………………………………..51 Structural evidence that the YA/HeJ and YWt chromosomes are different...52 Behavioral differences between the YA/HeJ and YWt chromosomes……...53 Why does the YA/HeJ cause disturbances in sexual differentiation?...........53 Acknowledgements………………………………………………………………58 Chapter 3: Disinfectants and Dysfunction: Solving the Mystery…………………..59 Abstract…………………………………………………………………………..60 Introduction………………………………………………………………………61 Methods and Materials…………………………………………………………...62 Breeding Protocols……………………………………………………….62 Superovulation…………………………………………………………...62 Methanol Cage Extractions………………………………………………62 Analytical Methods………………………………………………………63 Results…………………………………………………………………………....64 Problems in a new breeding colony are immediately apparent………….64 Ruling out the obvious candidates……………………………………….65 New research studies reveal continued reproductive disturbances………67 vii Are we victims of our own research?........................................................70 Discontinued use of the disinfectant but persistence of the chemical compounds……………………………………………………………….72 Inadvertent recontamination……………………………………………..73 Discussion………………………………………………………………………..77 Chapter 4: Summary and Future Directions………………..……………………….80 Summary…………………………………………………………………………80 The Mouse A/HeJ Y Chromosome: Another Good Y Gone Bad……….80 The A/HeJ Y Chromosome Induces Hermaphroditism………… 80 Comparison on A/HeJ and BALB/cWt…………………………..81 Sequence…………………………………………………81 Segregation Behavior…………………………………….82 Structural Analysis of the YA/HeJ Chromosome………………….83 Morphological Aberration of the A/HeJ Y Centromere…………83 Disinfectants and Dysfunction: Solving the Mystery…………………...84 Breeding Problems in the New Colony………………………….84 Changing the Environment………………………………………84 Reproductive Problems Continue………………………………..85 Identifying the Source of the Reproductive Disturbances……….85 Eliminating the Quaternary Ammonium Disinfectant…………...86 Accidental Recontamination……………………………………..87 Future Directions………………………………………………………...88 viii Studies of the A/HeJ Y Chromosome……………………………88 Centromere……………………………..………………...88 Sexual Differentiation…………………….……...………89 Epigenetic Analysis………………………...……………91 Analysis of the Effect of Quaternary Ammonium Compounds on Reproduction……………………………………………..92 Bibliography…………………………………………………………………………….95 ix List of Tables Chapter 1 Table 1.1 Examples of proteins that localize to the centromere and kinetochore and their functions…………….……………………………………………………………11 Table 1.2 Examples of studies of the impact of chemical exposure on human fertility...21 Table 1.3 Examples of common quaternary ammonium compounds and the types of products typically containing each one………………………………………….26 Chapter 2 Table 2.1 Analysis of Y chromosome hyperploidy and gaps…………………….…......42 Table 2.2 Meiotic analysis in C57BL/6J-YA/HeJ males…………………………….…….47 x List of Figures Chapter 1 Figure 1.2 Regions of the centromere and kinetochore………………………………....12 Chapter 2 Figure 2.1 Southern analysis of Bkm repeats in genomic DNA………………………...40 Figure 2.2 The frequency of Y chromosome hyperploidy and of chromatid gaps are not correlated in individual C57BL/6-YA/HeJ animals……………………44 Figure 2.3 Representative images of YA/HeJ chromosome morphological aberrations observed during analysis of A/HeJ Y chromosome in liver and fibroblast cells…………………………………………………………………………..45 Figure 2.4 Schematic showing the normal localization and a brief description of the eight FISH probes used in the structural analysis of the A/HeJ Y chromosome…..49 Figure 2.5 Structural analysis of the A/HeJ Y chromosome……………………………50 Chapter 3 Figure 3.1 A morphologically abnormal 18.5 day A/J fetus…………………….………66 Figure 3.2 Pregnancy rates in young (6-8 week) virgin C57BL/6J females………….…68 Figure 3.3 Cage extracts reveal contamination with quaternary ammonium compounds……………………………………………………………….…..73 Figure 3.4 Quaternary ammonium contaminants are transferred during cage washing...76 xi Investigation of Factors Affecting Fertility: Chromosome Segregation Errors and Environmental Toxins Abstract by JODI M. JACKSON If a species cannot propagate, it cannot survive. Human fertility is thought to be declining, with a myriad of possible causes. The objective of this dissertation was to investigate two factors that affect fertility: chromosome segregation errors and environmental toxins. Aneuploidy affects 10-30% of human conceptions and is the leading genetic cause of pregnancy loss and mental retardation. Accordingly, the etiology of chromosome segregation errors has long been studied. Conversely, while examples of environmental toxins impacting fertility can be found throughout the literature, this field is only recently becoming a major focus of research. The studies done for this dissertation were done independently from one another and are presented here separately. The observation of hermaphroditic mice on the inbred strain A/HeJ prompted the investigation of the YA/HeJ chromosome. The YA/HeJ chromosome was transferred to the C57BL/6J background and the hermaphroditism trait segregated with it, indicating the two are linked. We found the YA/HeJ chromosome to be prone to missegregation, however, we did not find evidence that segregation errors cause the disturbances in xii sexual differentiation. Interestingly, we frequently observed a gap between YA/HeJ sister chromatids, indicating inappropriate centromere function. The mouse Y centromere is near the