Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2019 Deciphering the ovarian proteomic impacts of obesity Kendra Leah Clark Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Genetics Commons Recommended Citation Clark, Kendra Leah, "Deciphering the ovarian proteomic impacts of obesity" (2019). Graduate Theses and Dissertations. 17659. https://lib.dr.iastate.edu/etd/17659 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Deciphering the ovarian proteomic impacts of obesity by Kendra Leah Clark A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Genetics and Genomics Program of Study Committee: Aileen F. Keating, Major Professor Jeffrey J. Essner Donald S. Sakaguchi Jason W. Ross Geetu Tuteja The student author, whose presentation of the scholarship herein was approved by the program of study committee, is solely responsible for the content of this dissertation. The Graduate College will ensure this dissertation is globally accessible and will not permit alterations after a degree is conferred. Iowa State University Ames, Iowa 2019 Copyright © Kendra Leah Clark, 2019. All rights reserved. ii DEDICATION I dedicate this thesis to my children; whose presence gave me reason. Additionally, I dedicate this work to all the people out there with questionable pasts that everyone gave up on but who never gave up on themselves. iii TABLE OF CONTENTS Page ABSTRACT vi CHAPTER 1. GENERAL INTRODUCTION 1 Dissertation Organization 1 Ovarian development, structure, and function 1 Folliculogenesis 3 Steroidogenesis 6 Hypothalamic pituitary gonadal axis 8 Ovarian gap junctions 10 Obesity 13 Gestational diabetes mellitus 15 Chemical-induced ovotoxicity 16 Chemotherapeutics 19 DNA damage response 21 Follicular apoptosis and atresia 24 Summary 28 References 29 Figures and figure legends 46 CHAPTER 2. DEVELOPMENTAL ORIGINS OF OVARIAN DISORDER: IMPACT OF MATERNAL LEAN GESTATIONAL DIABETES ON THE OFFSPRING OVARIAN PROTEOME IN MICE 51 Abstract 51 Introduction 52 Materials and methods 54 Results 60 Discussion 65 References 71 Figures and figure legends 77 Supplemental figures and legends 84 CHAPTER 3. OBESITY INDUCES BASAL OVARIAN DNA DAMAGE AND REDUCES ATM PHOSPHORYLATION 97 Abstract 97 Introduction 98 Materials and methods 100 iv Results 104 Discussion 106 References 111 Figures and figure legends 117 Supplemental figures and legends 124 CHAPTER 4: ATAXIA TELANGIECTASIA MUTATED COORDINATES THE OVARIAN DNA REPAIR AND ATRESIA-INITIATING REPSONSE TO PHOSPHORAMIDE MUSTARD 128 Abstract 128 Introduction 129 Materials and methods 131 Results 137 Discussion 143 References 152 Figures and figure legends 161 Supplemental figures and legends 168 CHAPTER 5. LEAN MATERNAL GESTATIONAL DIABETES AND HYPERPHAGIA-INDUCED OBESITY IMPACTS OVARIAN GAP JUNCTION PROTEIN EXPRESSION 202 Abstract 202 Introduction 203 Materials and methods 206 Results 209 Discussion 210 Conclusions 214 Declaration of interest 215 Funding 215 Author contribution statement 215 References 215 Figures and figure legends 221 CHAPTER 6. GENERAL CONCLUSIONS 225 Dissertation Summary 225 Future outlook 229 Conclusion 230 References 231 v APPENDIX. ISOLATION AND CULTURE OF PRIMARY EMBRYONIC ZEBRAFISH NEURAL TISSUE 233 ACKNOWLEDGEMENTS 270 vi ABSTRACT The ovary is the female reproductive organ responsible for the production of both the female gamete, the oocyte, and two major female sex hormones, estradiol and progesterone. During embryonic development, oocytes are formed from primordial germ cells and eventually become surrounded by squamous granulosa cells in a follicular structure, termed primordial. The oocyte numbers encased in primordial follicles are finite at birth and remain arrested in the diplotene stage of meiosis until ovulation or they degenerate through atresia. Once the pool of primordial follicles is depleted, ovarian senescence occurs. We hypothesized that the maternal metabolic changes that occur during lean gestational diabetes mellitus would impact offspring ovarian function both basally and in response to a dietary stressor later in life. We observed impacts on follicle numbers and alterations in the ovarian proteome, suggesting possible impacts on fertility and oocyte quality in relation to in utero and metabolic stressors. Additionally, we hypothesized that the ovarian DNA damage response is altered during obesity in adulthood. An elevated response in markers of DNA damage was observed, indicating that the metabolic status of the ovary during obesity initiates a low-level DNA damage response. Intercellular communication is also affected by a metabolic syndrome such as obesity or GDM exposure, with reduction of the gap junction protein Connexin-43 expression in antral follicles from ovaries that experienced obesity. Finally, to elucidate the molecular mechanisms behind the induction of the DNA damage response in the ovary after phosphoramide mustard exposure, we hypothesized that the DNA damage response would be blunted due to reduced abundance of the ATM protein. Using an Atm +/- mouse model to investigate impacts on folliculogenesis and the ovarian proteome, we determined that vii Atm haploinsufficiency results in an irregular DNA damage response, alters the ovarian proteome, and impacts the rate of follicle loss after phosphoramide mustard exposure. Taken together, these findings demonstrate that the DNA damage response is initiated in the ovary during times of metabolic stress and in the absence of Atm , unhealthy follicles remain in the ovary, potentially contributing to poor oocyte quality or infertility. 1 CHAPTER 1. GENERAL INTRODUCTION Dissertation Organization This dissertation includes four manuscripts relevant to this doctoral work, which have either been published or submitted for publication in peer-reviewed journals. These manuscripts establish the foundation of the dissertation work and are preceded by a general introduction describing the significance of the research topic and review of the literature, and proceeded by a discussion of general findings, conclusions, and future considerations. In addition, portions of the literature review section of the dissertation have been published as an invited review and an additional manuscript published outside of the scope of the dissertation is included in the appendix. Literature Review Parts of the literature review are adapted from: Clark KL , S Ganesan, AF Keating. 2018. Impact of toxicant exposures on ovarian gap junctions. Reprod. Toxicol . 81: 140-146. Ovarian development, structure, and function Early in mammalian embryonic development, the highly specialized primordial germ cells (PGCs), originate from the post implantation epiblast in response to extra- embryonic signaling from bone morphogenic proteins BMP4, BMP2, and BMP8B [1-3]. The proteins then bind with receptors that phosphorylate mothers against decapentaplegic 2 homologs SMAD1 and SMAD5, which then dimerize with SMAD4, leading to translocation into the nucleus to initiate the transcriptional regulators of the PCGs – PR domain zinc finger protein 1 (BLIMP1), PR domain zinc finger protein 14 (PRDM14), and transcription factor AP-2 gamma (AP2 γ) [4-6]. The PGCs then migrate from the posterior primitive streak into the endoderm, through the hindgut, and into the dorsal mesentery before arriving at the genital ridge [7, 8]. Once at the genital ridge, cell migration is complete and the PGCs associate with the somatic cells of the gonad and eventually acquire their respective sex-specific morphologies and functions. Some of these cells will serve as germline stem cells, which undergo meiosis to produce the female or male gametes. In the female, the PGCs divide synchronously with incomplete cytokinesis to form a cluster of cells connected by intracellular bridges, forming a multi-nucleated germ cell nest [9, 10]. Coordinated somatic cell migration and germ cell apoptosis contribute to the packaging of individual germ cell nuclei, which become surrounded by flattened pre-granulosa cells to form the finite primordial follicle pool [9, 10]. The early mammalian gonad is bipotential, composed of undifferentiated precursor cells that can follow one of two possible fates, male or female. The Wolffian (mesonephric) ducts are the progenitors for the male reproductive tract and the Müllerian (paramesonephric) ducts are the precursors of the female reproductive tract [11]. Sex determination is triggered by the expression and proper function of the testes determining gene sex-determining region Y ( Sry) in the presence of the Y chromosome, which signals cells to produce testosterone and anti-Müllerian hormone (AMH), resulting in the degeneration of the Müllerian ducts and differentiation of the testes [12, 13]. Conversely, the lack of Sry expression results in the regression of the Wolffian ducts and the 3 differentiation into the female reproductive tract [14]. The exact mechanisms behind early ovarian development are not well understood, though wingless-type MMTV integration site family member 4 ( Wnt4), nuclear receptor subfamily 0, group B, member 1 (Dax1), and forkhead