CHANGES IN NUCLEUS ACCUMBENS GENE EXPRESSION ACCOMPANY SEX-SPECIFIC SUPPRESSION OF VOLUNTARY PHYSICAL ACTIVITY IN AROMATASE KNOCKOUT MICE A Thesis Presented to the Faculty of the Graduate School at the University of Missouri, Columbia ¨ In partial fulfillment of the requirements for the Degree Master of Science By DUSTI SHAY Victoria Vieira-Potter, Thesis Supervisor JULY 2019 The undersigned, appointed by the dean of the Graduate School, have examined the thesis entitled Changes in Nucleus Accumbens Gene Expression Accompany Sex-Specific Suppression of Voluntary Physical Activity in Aromatase Knockout Mice Presented by Dusti Shay, a candidate for the degree of Master of Science, and hereby certify that, in their opinion, it is worthy of acceptance. _______________________________________ Dr. Victoria Vieira-Potter _______________________________________ Dr. Cheryl Rosenfeld _______________________________________ Dr. Dennis Lubahn _______________________________________ Dr. Mark Milanick _______________________________________ Dr. Jill Kanaley Dedicated to…… My beloved husband, my dear children, and the legacy that came before me. Acknowledgements From the moment I was accepted into the NEP department, I have been taken under the wing of multiple professors who have believed in me and invested in me to make me a better scientist. I have been so blessed to have Dr. Vieira-Potter as my primary mentor. Her support and encouragement have carried me through days when I didn’t believe in myself. Her guidance has pointed me in a positive direction in science, in self-care, and in life. Throughout my master’s program, Dr. Cheryl Rosenfeld has also been a mentor to me. I have been so grateful for her feedback, encouragement, and willingness to teach me and invest in me since I began my degree program. Without the help of these brilliant women, this master’s thesis would not be possible. I would also like to thank Dr. Dennis Lubahn for always being willing to talk science, teach me, and for his constant encouragement that I could, in fact, do the things I didn’t think I could do. I want to thank Dr. Jill Kanaley for being a consistent voice of reason as I have made choices on my career path. She has always provided me with an unbiased, straightforward opinion and I have always appreciated the clarity. I want to thank Dr. Mark Milanick for being a consistent role model for me over the last 9 years. Dr. Milanick has always been an inspiration to me to never make assumptions and always try to look at things from a different perspective. After teaching for him for 7 years, I knew that if I still loved physiology that much, then I could make it my career. I want to thank Dr. Kevin Fritsche, for asking me the right questions. It was his inspiration that led me to question my real motives for applying to the PhD program, and it was ultimately one of his statements that motivated me to continue toward a doctorate. ii I would like to give a huge thank you to everyone who has assisted me with this thesis project. Becky Welly, for being an amazingly organized and level-headed lab manager, and for allowing me to vent about grad school struggles, but never letting me whine too much. Stephanie Clookey and Terese Zidon, who helped teach me to be a grad student and who told me that what I was dealing with was completely normal. I want to thank Dr. Vieira-Potter’s, Dr. Rosenfled’s, and Dr. Lubahn’s lab teams for assisting with everything from data collection, to cage changes, to data analysis, and trouble-shooting when things didn’t quite work out. Julia Hartzigeorgiou, Brittney Marshall, Juide Mao, Michelle Farrington, Nathan Mahloch…none of this would have happened without you. Finally, I need acknowledge the love and support of my husband and family through this journey. I took on this endeavor and David has supported and encouraged me the whole way. We have fought battles together and come out on the other side a little bit stronger every time. I could not do this without him. I also need to thank my children, who have done their best to understand that they are my reasons for following this path. I want to show my children that they can accomplish anything, no matter how hard, and that they can be unafraid to fail, because true failure only happens when you quit trying. iii Table of Contents Acknowledgements………………….…………..……………………………………….ii List of Figures……………………….…………...……………………………………… v List of Tables ……………………………………………………………………………vi Abstract………………………………...……………………………………………….vii Chapter 1-Literature Review, Rationale, & Specific Aims……………………………1 Chapter 2-Effect of aromatase ablation on phenotype and NAc……………………33 gene expression in male and female mice. 1. Introduction………………………………………………………………33 2. Materials and Methods…………………………………………………...35 3. Results……………………………………………………………………44 4. Discussion………………………………………………………………..72 Chapter 3- Study Limitations and Future Directions………………………………..78 References…………………………………………………………………………….....80 iv List of Figures Figure 1: Highlighted Tissues that Express Aromatase in Humans………………………1 Figure 2: Synthesis of Estradiol from Testosterone……………………………………….2 Figure 3: Aromatase ablation significantly increases fat mass in females………………45 Figure 4: Aromatase ablation promotes a positive energy balance in females only via…47 reductions in physical activity Figure 5: Aromatase ablation alters sleep duration in females only, but has no effect….49 on anxiety-like or learning behavior in either sex. Figure 6a: Sex differences in NAc gene expression……………………………………..52 Figure 6b: Genotype differences in Nac gene expression……………………………….56 Figure 7a: Correlation matrix across all mice……………………………………………59 Figure 7b: Correlation matrix in female mice only……………………………………...62 Figure 8: STRING PPI Interactions……………………………………………………...64 v List of Tables Table of Contents…………………………………………………………………………iv Table 1: Primer sequences used for NAc q-pcr………………………………………….43 Table 2: Description of reads from RNAseq data in Nac ……………………………….50 Table 3: Gene descriptions of top 30 DEGs between male and female WT & ArKO mice..…………………………………………………..………………….53 Table 4: Gene descriptions of top 30 DEGs between WT & ArKO male and female mice ……………………………………..…………………………….57 Table 5: Significantly enriched interactions of DEGs indicating the gene ontology……66 and biological pathways (KEGG & Reactome) in WT males v. WT females Table 6: Significantly enriched interactions of DEGs indicating the gene ontology……68 and biological pathways (KEGG & Reactome) in KO males v. KO females Table 7: Significantly enriched interactions of DEGs indicating the gene ontology……70 and biological pathways (KEGG & Reactome) in Male WT v. ArKO mice Table 8: Significantly enriched interactions of DEGs indicating the gene ontology……71 and biological pathways (KEGG & Reactome) in Female WT v. ArKO mice vi Changes in Nucleus Accumbens Gene Expression Accompany Sex-Specific Suppression of Voluntary Physical Activity in Aromatase Knockout Mice Dusti A. Shay Dr. Victoria Vieira-Potter, Thesis Mentor Abstract Aromatase is the rate-limiting enzyme in the conversion of testosterone to estrogen and is highly conserved across species. Post-menopause, the aromatase enzyme (locally expressed in adipose tissue, bone, breast tissue, and brain) is the primary source of estradiol in females, as well as the sole source of estradiol in males throughout the lifespan. Notably, secondary sources of estrogens (e.g. phytoestrogens, 27- hydroxycholesterol) also bind estrogen receptors alpha and beta, but with lower affinity (Starkey, Li et al. 2018, Vieira-Potter, Cross et al. 2018). Loss of estrogen contributes to metabolic dysfunction, primarily in women, resulting in obesity and insulin resistance. Pharmacologic downregulation of aromatase activity via aromatase inhibitors is commonly used in males and females for treatment of diseases associated with estrogen (e.g. short stature in pubertal boys or adjuvant hormone therapy in postmenopausal breast cancer). However, the implications of suppression of aromatase on neurologic functions, such as cognition, memory, and mood, and even motivation for physical activity in the nucleus accumbens (NAc) brain region via alterations in dopamine signaling, are not vii entirely clear. Previous work using an aromatase knockout mouse model has established that female knockout mice experience a reduction in physical activity, but sex comparisons have not been made. Objective: An aromatase knockout (ArKO) mouse model was used to evaluate sex differences in both phenotype and gene expression in the nucleus accumbens. We hypothesized that physical activity would be significantly depressed in ArKO females but not ArKO males. Additionally, we postulated that NAc gene expression patterns would be differentially regulated by sex and correlate with the degree of physical inactivity. Methods: Male and female wild-type and ArKO mice were compared for body weight, body composition (EchoMRI), energy intake/expenditure (MET chambers), and behaviors such as: sleep, spontaneous physical activity (MET chambers), spatial memory (Barnes maze), and anxiety-like behavior (Elevated Plus Maze). Gene expression in the nucleus accumbens brain region were analyzed via RNAseq comparisons and correlated to phenotypic traits. Finally, protein- protein interactions and pathway enrichment of differentially expressed genes were performed. Results: Female ArKO mice (n = 9) have a greater percent body fat (WT=14.13% ± 4.1 v. ArKO=9.45% ± 1.6; p < 0.05) and lower percent lean mass (WT=83.6%