A Dissertation entitled Hypothalamic Melanocortin 4 Receptors Regulate Sexual Behavior in Mice by Erin Semple Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biomedical Sciences ________________________________________ Jennifer Hill, PhD, Committee Chair ________________________________________ David Giovannucci, PhD, Committee Member ________________________________________ Joshua Park, PhD, Committee Member ________________________________________ Edwin Sanchez, PhD, Committee Member ________________________________________ Ruili Xie, PhD, Committee Member ________________________________________ Amanda Bryant-Friedrich, Dr. rer Nat., Dean College of Graduate Studies The University of Toledo August 2017 Copyright 2017, Erin A. Semple This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of Hypothalamic Melanocortin 4 Receptors Regulate Sexual Behavior in Mice by Erin Semple Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biomedical Sciences The University of Toledo August 2017 Sexual dysfunction affects approximately one third of men and 40% of women in the United States and world-wide. Unfortunately, existing treatments that target the periphery are not effective for all types of sexual dysfunction, and the involvement of the central nervous system in sexual behavior is not well-understood. Preclinical data has shown that central melanocortins and their receptors, particularly the melanocortin 4 receptor (MC4R), may play a role in sexual behavior in both male and female rodents. We found that six-month-old male MC4R null mice showed evidence of erectile dysfunction and an inability to ejaculate. Due to the high expression of MC4R in the paraventricular nucleus of the hypothalamus (PVN), we hypothesized that the PVN is a key site of melanocortin-mediated sexual behavior. Using Sim1 as a target for the PVN, we tested the sexual behavior of a tbMC4Rsim1 transgenic mouse model in which MC4R was only expressed on Sim1 neurons. These mice did not have the sexual impairments seen in MC4RKO mice, implying that MC4R on Sim1 neurons is sufficient for erectile function and ejaculation. To reduce the confound of age-related obesity in MC4RKO mice, we also tested these mice at two-months of age. The younger MC4RKO mice had a different phenotype, with the only sexual deficit being delayed ejaculation. This was also iii recovered in tbMC4Rsim1 mice. Furthermore, expressing MC4R only on oxytocin neurons similarly recovered the sexual impairment in MC4R null males. Finally, a metabolic profile indicated that the delayed ejaculation seen in MC4RKO mice at two months of age was independent of MC4R-mediated weight gain. Female MC4RKO mice had a decreased lordosis quotient at two months of age. Expression of MC4R only on Sim1 neurons or oxytocin neurons resulted in a lordosis quotient comparable to controls, despite a similar metabolic profile to MC4R null mice. This study implicates MC4R on Sim1 neurons, and more specifically oxytocin neurons, in the central neurocircuitry underlying sexual behavior in both male and female mice. iv Acknowledgements I would like to acknowledge my advisor, Dr. Jennifer Hill, for providing me with the opportunity to work in her lab. Without her guidance and support, none of this research would have been possible. I am also grateful for the constant advice, support, and companionship of the members of the Hill lab who made my graduate studies enjoyable. I would also like to thank my committee members, the faculty, staff, and students of the Department of Neurosciences as well as the Department of Physiology and Pharmacology, and my fellow MD/PhD students who have provided me with a nurturing environment throughout my time in graduate school. Finally, I would like to acknowledge my family and friends whose constant support and encouragement was invaluable. I owe a special shout-out to my significant other, Bradley, for having my back through all of the highs and lows of both medical school and graduate school. Table of Contents Abstract .............................................................................................................................. iii Acknowledgements ..............................................................................................................v Table of Contents ............................................................................................................... vi List of Figures .................................................................................................................. viii List of Abbreviations ...........................................................................................................x 1 Challenges in Understanding Sexual Dysfunction ..................................................1 1.1 Prevalence of Sexual Dysfunction ....................................................................1 1.2 Challenges in Definition ...................................................................................3 1.2.1 Definitions for Men.............................................................................3 1.2.2 Definitions for Women .......................................................................5 1.3 Comorbidities ....................................................................................................6 1.4 Treatment Options ............................................................................................8 1.3.1 Treatments for Men.............................................................................8 1.3.2 Treatments for Women .......................................................................9 1.5 Animal Models of Sexual Dysfunction...........................................................10 1.6 Central Control of Sexual Function ................................................................12 1.7 The Melanocortin System and Sexual Dysfunction........................................13 1.8 Study Objectives .............................................................................................15 v 2 Sim1 Neurons are Sufficient for MC4R-Mediated Sexual Function in Six-Month- Old Male Mice .......................................................................................................18 2.1 Introduction .....................................................................................................18 2.2 Methods….......................................................................................................20 2.3 Results….. .......................................................................................................23 2.4 Discussion .......................................................................................................27 2.5 Figures….........................................................................................................33 3 Hypothalamic Melanocortin 4 Receptors Influence Male Sexual Behavior in Young Mice ........................................................................................38 3.1 Introduction .....................................................................................................38 3.2 Methods….......................................................................................................40 3.3 Results….. .......................................................................................................44 3.4 Discussion .......................................................................................................51 3.5 Figures….........................................................................................................56 4 Hypothalamic Melanocortin 4 Receptors Affect Lordosis in Female Mice Independent of Metabolic Changes .......................................................................70 4.1 Introduction .....................................................................................................70 4.2 Methods….......................................................................................................72 4.3 Results….. .......................................................................................................76 4.4 Discussion .......................................................................................................79 4.5 Figures….........................................................................................................84 5 Summary of Findings and Future Directions .........................................................94 References ..........................................................................................................................99 vi Chapter 1 ................................................................................................................99 Chapter 2 ..............................................................................................................109 Chapter 3 ..............................................................................................................113 Chapter 4 ..............................................................................................................118 Chapter 5 ..............................................................................................................124 vii List of Figures 2-1 Sexual behavior in MC4RKO and tbMC4Rsim1 mice