Western University Scholarship@Western Electronic Thesis and Dissertation Repository 7-11-2013 12:00 AM The Role of Kisspeptin and KNDy Cells in the Reproductive Neuroendocrine System Christina M. Merkley The University of Western Ontario Supervisor Michael N. Lehman The University of Western Ontario Graduate Program in Neuroscience A thesis submitted in partial fulfillment of the equirr ements for the degree in Doctor of Philosophy © Christina M. Merkley 2013 Follow this and additional works at: https://ir.lib.uwo.ca/etd Part of the Endocrine System Commons, and the Neurosciences Commons Recommended Citation Merkley, Christina M., "The Role of Kisspeptin and KNDy Cells in the Reproductive Neuroendocrine System" (2013). Electronic Thesis and Dissertation Repository. 1386. https://ir.lib.uwo.ca/etd/1386 This Dissertation/Thesis is brought to you for free and open access by Scholarship@Western. It has been accepted for inclusion in Electronic Thesis and Dissertation Repository by an authorized administrator of Scholarship@Western. For more information, please contact [email protected]. THE ROLE OF KISSPEPTIN AND KNDY CELLS IN THE REPRODUCTIVE NEUROENDOCRINE SYSTEM by Christina M. Merkley Graduate Program in Neuroscience A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy The School of Graduate and Postdoctoral Studies The University of Western Ontario London, Ontario, Canada © Christina M. Merkley 2013 ABSTRACT The gonadotropin-releasing hormone (GnRH) neurons in the hypothalamus represent the final common output in the central control of reproduction. GnRH secretion is modulated indirectly by steroid feedback action of gonadal steroids on afferent interneurons. In recent years, the neuropeptide, kisspeptin, has emerged as a key mediator of steroid feedback onto GnRH neurons and the reproductive system. Kisspeptin neurons located in the preoptic area (POA) mediate estradiol (E2) positive feedback leading to the preovulatory surge, and kisspeptin cells in the arcuate nucleus (ARC) have been shown to mediate the negative feedback actions of E2 on pulsatile GnRH secretion. ARC kisspeptin neurons are distinguished from those in POA by their extensive reciprocal connections with each other, and their co-expression of two other neuropeptides, neurokinin B (NKB) and dynorphin. Because of this co-expression, the ARC kisspeptin neurons have been termed “KNDy” (Kisspeptin/NKB/Dynorphin) cells. Using sheep and mice as neuroendocrine models, the goal of this dissertation was to investigate the functional organization of KNDy cells and their projections to GnRH neurons, in order to gain insight into their role as mediators of steroid feedback. First, we showed that, contrary to expectations based on rodent work, KNDy neurons in the sheep are activated during both positive and negative feedback of E2 suggesting that they are common mediators of both GnRH pulses and the GnRH surge. Next, using KNDy cell peptides as markers, we showed that KNDy neurons in the sheep send direct neuronal projections to a majority of GnRH cell bodies spread over multiple regions, as well as input to GnRH terminals in the median eminence. We then explored the potential plasticity of synaptic inputs onto KNDy and GnRH neurons across the ovine estrous cycle, and found a significant increase in the total number of inputs to KNDy neurons, as well as KNDy inputs to GnRH cells, at the time of the preovulatory GnRH surge. Performing similar experiments in transgenic mice, we tested the hypothesis that plasticity in these inputs is regulated, in part, by E2. Finally, we began to investigate the possibility that NKB released by KNDy cells might play a functional role in the GnRH surge, by examining internalization of the NKB receptor, NK3R, across the estrous cycle. Taken together, these findings provided novel information on the roles of kisspeptin and KNDy neurons in steroid feedback control of GnRH secretion, and set the stage for future experiments to ii explore the mechanisms for steroid feedback in individual neurons, the functional role of synaptic plasticity in this system, and the role of KNDy peptides in regulating the reproductive neuroendocrine system across the estrous cycle. Keywords: Kisspeptin, KNDy, estrous cycle, estradiol, neural activation, gonadotropin- releasing hormone (GnRH), reproduction, luteinizing hormone, glutamate, neurokinin B, dynorphin, confocal microscopy, immunohistochemistry, immunofluorescence, plasticity, neuroendocrinology, hypothalamic-pituitary-gonadal axis. iii CO-AUTHORSHIP All contents of the current dissertation were carried out in collaboration with Michael N. Lehman and Lique M. Coolen. A large majority of this work (chapters 2-5) is also in collaboration with Robert L. Goodman, long-time collaborator of Drs. Lehman and Coolen. Chapter 2 was co-written by Christina M. Merkley and Michael N. Lehman with inputs by Robert L. Goodman and Lique M. Coolen. Chapter 3 was written by Christina M. Merkley and Katrina L. Porter, with intellectual inputs by Stan M. Hileman, Robert L. Goodman, Michael N. Lehman and Lique M. Coolen. Animal procedures and tissue collection (sections 3.2.1-2.2.4) were conducted at West Virginia University under the supervision of Robert L. Goodman. Experimental procedures and data analyses (section 3.2.5-3.2.6) were performed by Christina M. Merkley for Experiment 1, and Katrina L. Porter for Experiment 2. Chapter 4 was written by Christina M. Merkley, with intellectual inputs by Robert L. Goodman, Lique M. Coolen and Michael N. Lehman. Animal procedures and tissue collection (sections 4.2.1-4.2.2) were carried out at West Virginia University under the supervision of Robert L. Goodman. All experimental procedures, confocal microscopy, and data analysis (sections 4.2.3-4.2.5) were performed by Christina M. Merkley. Chapter 5 was written by Christina M. Merkley, with intellectual inputs by Robert L. Goodman, Lique M. Coolen and Michael N. Lehman. Animal procedures (sections 5.2.1-5.2.4) were conducted at West Virginia University, while experimental procedures, confocal microscopy and data analysis (sections 5.2.5- 5.2.7) were performed out by Christina M. Merkley with inputs by Lique M. Coolen. Chapter 6 was written by Christina M. Merkley, with intellectual inputs by Michael N. Lehman. Animal procedures (sections 6.2.1- 6.2.2) were carried out at the University of Texas Southwestern Medical Center under the supervision of Carol F. Elias. Experimental procedures, confocal microscopy and data analysis (sections 6.2.3-6.2.6) were performed by Christina M. Merkley. Chapter 7 was written by Christina M. Merkley with intellectual inputs by Michael N. Lehman. Animal procedures (sections 7.2.1-7.2.3) were conducted at Texas A&M University under the supervision of Marcel Amstalden. Experimental procedures, confocal microscopy and data analysis was conducted by Christina M. Merkley (sections 7.2.4-7.2.5) with inputs by Lique M. Coolen. iv In addition to the work described above, Christina M. Merkley has taken part in animal work at the Sheep Research Facility at the University of Michigan, Ann Arbor, Michigan, and has contributed to numerous research studies at this facility. This includes perfusions and brain extractions, blood sampling, blood processing, fetal tissue collection surgery, fetal organ preservation, and lambing (ear tagging, weighing and blood sampling). v ACKNOWLEDGEMENTS First, I would like to acknowledge my supervisor, Dr. Mike Lehman for his support and encouragement throughout the years. Thank you for believing in me from the start, having patience with me, and giving me every opportunity to make the best of these five years. Dr. Lehman has been an incredible mentor, and has given me opportunities to present my work all over the world. I would also like to thank Dr. Lique Coolen for her advice and guidance. She has a solution to almost every problem that comes up in the lab, and through daily supervision has been a big part of my successes. I would like to thank Dr. Bob Goodman at West Virginia University, who has spent a lot of time editing and revising my manuscripts over the years, and providing constructive guidance. I would also like to thank the members of my advisory committee, Dr. Andy Babwah and Dr. Raj Rajakumar for their encouragement, guidance and support. I would like to thank the members of the Coolen-Lehman Lab and the Goodman team. I have learned about what collaborating and working together really means when it comes to accomplishing goals. It is so inspiring to be surrounded by people who are both colleagues and friends. I would like to acknowledge my wonderful and loving family for their support and encouragement. Thank you for believing in me, and teaching me about what hard work, commitment, and perseverance really mean. In addition, I would like to acknowledge my fiancé Peter de Jonge, who has been a source of such great joy for me. Thank you for your unending love, understanding and patience. I am so lucky and blessed to have you in my life. Finally, I would like to extend thanks to the many other people who have made this journey so memorable for me. This includes friends and colleagues in Ann Arbor, Michigan, and the members of Briarwood Presbyterian Church in Jackson, Mississippi. Thank you all for your support, encouragement and inspiration. vi TABLE OF CONTENTS TITLE I ABSTRACT II CO-AUTHORSHIP IV ACKNOWLEDGEMENTS VI LIST OF TABLES XII LIST OF FIGURES XIII LIST OF APPENDICES XVI LIST OF ABBREVIATIONS XVII CHAPTER 1: GENERAL INTRODUCTION 1 1.1. Introduction 2 1.1.1. The Reproductive Axis 2 1.1.2. Modes of GnRH Secretion and their Control by Steroid Hormones 2 1.2. Steroid Feedback Control of GnRH Neurons: Role of Kisspeptin 6 1.2.1. Kisspeptin 8 1.2.2. Kisspeptin and Reproduction 8 1.2.3. Stimulatory Effects of Kisspeptin on Gonadotropin Secretion in Mammals 10 1.2.4. Actions of Kisspeptin on GnRH Neurons 10 1.2.5.