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% ± 1.9 v. ArKO=78.4% ± 3.1; p < 0.05) compared to WT females (n = 11).

Wild-type (n = 8) and ArKO (n = 10) males do not differ significantly in these measures.

ArKO females consume less energy (WT=0.60 ± 0.23 kcal/g BW/day v. ArKO=0.46 ±

0.06 kcal/g BW/day; p < 0.05) than WT females and have a lower resting energy expenditure than WT females (WT=0.014±0.0008 kcal/g BW/day v. ArKO=0.011±0.001 kcal/g BW/day; p < 0.05), similar to WT males when expressed relative to body weight.

The primary difference in total energy expenditure in ArKO females can be contributed

viii to the significant decreases in spontaneous physical activity such that they are even less active than WT males, since reductions in REE are not significant enough to entirely explain the obesity phenotype. ArKO females also sleep more during the night cycle when mice are primarily active (WT=4.83±0.76 hrs/12 hr. cycle v. ArKO=8.19±1.6 hrs/12 hr cycle; p < 0.05). NAc gene expression (Per3, Pts, Layn, Dixdc1, Cryab, Nxpe4, and Gldn) correlates strongly with spontaneous physical activity (24 hrs) (R = -0.81,

0.88, 085, 0.85, 0.70, 0.76, & 0.69 respectively), sleep during the dark cycle (R = 0.66, -

0.87, -0.83, -0.75, -0.78, -0.68, & -0.73 respectively) and relative energy intake

(R = -0.81, 0.88, 0.86, 0.85, 0.70, 0.76, 0.69 respectively) specifically in females.

Conclusion: Aromatase ablation contributes to an obesity phenotype in both sexes, but more so in females, primarily due to reductions in physical activity. NAc gene expression correlates strongly with behaviors that also contribute to obesity, specifically in females.

ix Chapter 1-Literature Review, Rationale, & Specific Aims

Introduction

Traditionally, estrogen (E2) has been considered a strictly-female, reproductive hormone, but research over the last few decades has shown that estrogen signaling is also important in males and has physiological effects far beyond just reproduction, including metabolic and cardiovascular effects, as well as cognition and neurobehavioral traits (Butler, Warden et al. 2010, Meng, Ni et al. 2011, Nugent, Wright et al. 2015, Vierk, Bayer et al. 2015, Iorga, Cunningham et al. 2017, Ohlsson, Hammarstedt et al. 2017). It is also true, that the gonads were once considered the primary source of estrogen physiologically, but research since the 1970s

(Naftolin, Ryan et al. 1972,

Naftolin, Ryan et al. 1975, Simpson,

Zhao et al. 1997) has discovered that the aromatase enzyme, the rate- limiting step in the conversion of https://picswe.net/pics/human-life-sciences-9f.html androgens to estrogen, is also present in adipose tissue, bone, and brain, and that local estrogen production via aromatase, even after the loss of gonadal hormones, also plays a significant role in estrogen’s actions (Simpson 2000, Ohlsson,

Hammarstedt et al. 2017) (Fig. 1). This review will provide a general overview of the role of the aromatase enzyme in various tissues of differing species, with specific focus on its function in the nucleus accumbens (NAc) brain region in

1 both animal models and humans and the implications of affecting aromatase activity in the brain.

Aromatase in the Brain – History and Overview

Aromatase is the rate-limiting enzyme in the irreversible conversion of C19 androgens to C18 estrogen (E2) in gonadal and extragonadal tissues (e.g. bone, adipose tissue, and brain) (Jones, Thorburn et al. 2000, Hero, Norjavaara et al.

2005, Blakemore and Thornton, M.J., et al., 2006 Naftolin 2016, Patel 2017,

Halper, Sanchez et al.

2019) (Fig. 2) (Thornton,

Nelson et al. 2006). This enzyme is required for estrogen synthesis in males Figure 2: Synthesis of estradiol from testosterone and females of various mammalian and non-mammalian species throughout the lifespan (Simpson, Clyne et al. 2002, Patel 2017). In fact, many mammalian species, including humans, utilize extra-gonadal aromatase as the sole source of estrogen in males of all ages and the primary source of estrogen in females after loss of ovarian hormones (Nelson and Bulun 2001). Naftolin et al. (Naftolin,

Ryan et al. 1971, Naftolin, Ryan et al. 1972) first discovered aromatase in the hypothalamus of the human fetus and then the rat brain, leading to the

“aromatization hypothesis” , claiming that aromatase in the brain is responsible for full masculinization of sexually dimorphic brain regions during fetal development (Naftolin, Ryan et al. 1972, Wright, Schwarz et al. 2010). Today, it

2 is understood that androgens and estrogens work in a synergistic manner for full masculinization and defeminization of the brain (Murray, Hien et al. 2009,

Kurian, Olesen et al. 2010, Schwarz, Nugent et al. 2010, Rosenfeld 2017). In addition, other genetic and epigenetic influences alter the physiological development of certain brain regions, including the hypothalamus, amygdala, and preoptic area (Murray, Hien et al. 2009, Kurian, Olesen et al. 2010, Schwarz,

Nugent et al. 2010, Matsuda, Mori et al. 2011).

Aromatase in the Human Brain

Studies of aromatase in the human brain were traditionally limited due to the availability of human brain tissue, such as Naftolin’s discovery of aromatase in the human fetus (Naftolin, Ryan et al. 1972). Today, measurements of aromatase in human brain and in vivo measures in animals are possible via PET imaging techniques by utilizing fluorescent labels for the aromatase protein to determine the brain areas in which aromatase is most active (Takahashi, Hosoya et al. 2014). Given these techniques and other studies identifying aromatase in the brain of humans, regions containing aromatase mRNA have been identified, including: hippocampus, amygdala, hypothalamus, frontal cortex, temporal lobe, thalamus, and inferior olive of the medulla (Sasano, Takashashi et al. 1998,

Stoffel-Wagner, Watzka et al. 1999, Yague, Munoz et al. 2006, Biegon, Kim et al.

2010). Although no sex differences have been identified to date, trends in the pituitary have been observed with males having higher expression of aromatase than females (Kadioglu, Oral et al. 2008).

3 In humans, congenital aromatase deficiency has only been reported in 15 people (8 men, 7 women) (Boon, Chow et al. 2010). In females, it is often diagnosed at birth as they present with pseudo-hermaphroditism and then primary amenorrhea, virilization, and failure of breast development at puberty, as well as struggles with fertility in adulthood (Lein, Hawrylycz et al. 2007, Boon, Chow et al. 2010). In males, diagnosis often occurs at puberty due to tall stature, delayed fusion of epiphyseal growth plates, and excess adiposity (Lein, Hawrylycz et al.

2007, Boon, Chow et al. 2010). In both sexes, supplemental E2 reverses symptoms (Lein, Hawrylycz et al. 2007, Boon, Chow et al. 2010).

Congenital aromatase deficiency is rare in humans (Jones, Boon et al.

2006, Bulun 2014), but pharmacologic manipulation of aromatase is common, often with the use of aromatase inhibitors in males and females of all ages (Hero,

Maury et al. 2010, Gentry, Erickson et al. 2018, Huang, Du et al. 2018, Shuling,

Sie Kuei et al. 2019). Systemic inhibition of aromatase action is commonly used as treatment in diseases associated with estrogen in people of all ages, as well as in animal models for examination of aromatase’s role in physiology. Two common aromatase inhibitors (AI) utilized are Anastrozole and Letrozole, which are both used to downregulate aromatase activity systemically.

In males, pubertal boys are prescribed AI for short stature, precocious puberty, or gynecomastia (Hero, Maury et al. 2010, Russell and Grossmann

2019). In older males, AI can be used to treat primary hypogonadism and raise the ratio of testosterone to estrogen, but this may not be the most effective treatment (Russell and Grossmann 2019). In females, AI are most commonly

4 used as adjuvant therapy for postmenopausal breast cancer, or in the treatment of ovulation induction, endometriosis, and poly-cystic ovarian syndrome (2016).

Roles of Aromatase in the Brain Across Species

The aromatase enzyme is highly conserved across multiple mammalian and non-mammalian species and plays a vital role in fetal development, skeletal growth into adulthood, metabolism of adipose tissue, and various behaviors associated with specific brain regions. Sexual differentiation is arguably the most important role of aromatase as inhibition of this enzyme, both genetically and pharmacologically, affects sexually dimorphic physiological responses in various tissues (Naftolin, Ryan et al. 1972, Dalla, Antoniou et al. 2004, Cao, Duan et al.

2012, McCarthy, Nugent et al. 2017). Numerous studies have characterized the role of aromatase in biological function, and both genetic models and pharmacologic interventions such as aromatase inhibitors (AI), have been used to manipulate aromatase in the brain with results often differing between species, sex, brain region, and methodology of the study. In addition to sexual differentiation, other cognitive functions and neurobehavioral responses are affected by aromatase, such as, learning and cognition, cellular stress response, recovery from traumatic brain injury, and mental health disorders (e.g. anxiety, depression, and schizophrenia) and will be discussed below.

Sexual Differentiation-Organization of the Sexually Dimorphic Brain

Aromatase in the brain of non-mammalian species (e.g. birds and fish) is much higher than in mammals and plays a greater role in sexual development

(Hutchison 1993, Cao, Duan et al. 2012, Dickens, Balthazart et al. 2012). As

5 such, these animal models have been utilized to study the effects of aromatase in the brain and behavior within these species. For example, aromatase genes

(cyp19a1a and cyp19a1b) along with dmrt1 play a role in determining the sex of the rare minnow (Gobiocypris rarus), even changing the sex of fish in adulthood if expression is altered (Cao, Duan et al. 2012).

Estrogen’s role in sexual differentiation of mammals is complex and occurs both during fetal development, organizing sexually differentiated brain regions, and later during puberty, where it functions to activate typical adult sexual behaviors (Naftolin, Ryan et al. 1972, Dalla, Antoniou et al. 2004, Wright,

Schwarz et al. 2010). Organizational masculinization and defeminization of certain brain regions of various vertebrate species is thought to depend on the aromatization of testosterone to estradiol during a critical period of fetal development which varies among species (Dalla, Antoniou et al. 2004, Harada,

Wakatsuki et al. 2009, McCarthy, Nugent et al. 2017). For instance, brain regions associated with sexual differentiation and behavior (e.g. hypothalamus and medial preoptic area (MPOA)) depend on the presence of sex steroids for masculinization or feminization, while other brain regions express aromatase, but do not change expression or aromatase activity after removal of E2 or testosterone (T)

(Abdelgadir, Resko et al. 1994, Naftolin 1994, Roselli, Stormshak et al. 1998).

During the early neonatal period, a surge in gonadal hormones exposes the male brain to more hormones (testosterone, DHT, and E2) than females, driving the organizational process of sexual differentiation. Estrogen works in this process mainly through ERa, which is lower in males than in females within the preoptic

6 area (POA), and negatively correlates with the level of methylation of the ERa exon 1b promoter. (Kurian, Olesen et al. 2010). It is also important to note that maternal estrogens are not affecting fetal sexual differentiation due to the presence of alpha fetoprotein, without which, maternal estrogens would masculinize and defeminize the developing fetal brain, resulting in disruptions to the hypothalamic-pituitary axis and often issues with fertility (Bakker, De Mees et al. 2006, De Mees, Laes et al. 2006).

Some evidence suggests that this critical window of development can extend into the postnatal period, providing epigenetic influence on mechanisms of sexual differentiation (Kurian, Olesen et al. 2010, Wright, Schwarz et al. 2010,

Nugent, Wright et al. 2015). Even after birth, exposure of female rats to estradiol within the first 2 postnatal days (similar to the heightened levels in males of the same age) increases methylation of the ESR1 promoter regions 1 & 6 specifically and decreases ESR1 mRNA expression in the POA of females, resulting in expression similar to males. (Kurian, Olesen et al. 2010). In addition, early rearing experiences can epigenetically alter sexually differentiated genes, leading to further sexually differentiated behaviors. For example, rat dams often groom the anogenital region of males more than females during the first week after birth, increasing methylation of the ERa promoter region in the MPOA of male pups, masculinizing later behaviors. In fact, similar grooming of female pups produces masculinized behaviors, and removal of this grooming in males lends to more feminized behaviors in adulthood. (Kurian, Olesen et al. 2010). While this process has been most fully investigated in rodents, within sheep, distribution of

7 ERa and androgen receptor (AR) mRNA in the sexually dimorphic nucleus of the medial preoptic area during the critical period of sexual differentiation overlaps with aromatase mRNA, suggesting that both androgens and estrogens play a role in the development of this brain region and may be responsible for sexually dimorphic development (Reddy, Estill et al. 2014). In other brain regions (e.g. the amygdala, medial basal hypothalamus, and frontal cortex), expression of ERa mRNA was higher in females, while AR mRNA expression did not differ between sexes in any region, suggesting a specific role of the aromatase enzyme

(Reddy, Estill et al. 2014).

Organizational roles of aromatase may also be affecting sexual differentiation by influencing gene expression (Harada, Wakatsuki et al. 2009).

Analysis of gene expression and pathway enrichment (KEGG) in the diencephalic regions of ArKO and WT mice (sex not specified) revealed that suppression of

JAK/STAT signaling pathways, oxidative phosphorylation pathways, and

SNARE vesicular transport pathways indicated that ArKO mice have phenotypic disruptions in proliferation/apoptosis and energy supply of neurons, insufficient transport of vesicles containing neurotransmitters, energy homeostasis, and apoptosis of dopaminergic neurons in the arcuate nucleus and MPOA (Harada,

Wakatsuki et al. 2009).

Sexual Differentiation-Activation of Socio-sexual Behaviors Postnatally

In addition to organization of the sexually dimorphic brain, the activation of sexual behaviors in adult animals (e.g. mounts, intromission, and ejaculation in male rodents and female behaviors such as lordosis and ovulation) is thought to

8 occur by the same mechanism as organization in terms of increased exposure to sex hormones (Dalla, Antoniou et al. 2004). This occurs centrally in the brain and does not affect circulating levels of estrogen, although increases in gonadal sex hormones also occur during this time period (Dalla, Antoniou et al. 2004). The extent to which disruption of this process affects reproductive behaviors varies by species (Dalla, Antoniou et al. 2004). In mice, male ArKO mice show disrupted coital behavior and olfactory investigation of volatile body odors of the opposite sex (Dalla, Antoniou et al. 2004). Even though coital behavior can be rescued with either exogenous estradiol or DHTP (a non-aromatizable form of testosterone), olfactory investigation of volatile body odors is not rescued (Dalla,

Antoniou et al. 2004). This data points to the differential organizational/activational patterns of estradiol in male rodent reproductive behavior.

Activational effects of aromatase are brain-region specific and may be different between species. For example, male Zebra finches have greater aromatase activity in the interconnected nuclei of the forebrain which contributes to courtship rituals and learning songs during development (Wade 2001).

Japanese quail have been studied quite commonly among bird species, especially with regards to the effects of stress on aromatase activity in specific brain areas controlling sexual behavior. In the quail brain, stress induces an increase in aromatase activity in the medial preoptic nucleus, whereas sexual behavior drives aromatase activity down. In male quail, these changes in brain aromatase activity are reduced within 5 minutes of sexual activity. In addition, male sexual behavior

9 does not change if stress is induced prior to contact with a female, nor is their behavior influenced if their female partner is stressed prior to sexual contact.

However, stress-induced increases in aromatase activity in the medial preoptic area of female quail are not rescued with partner pairing, and female sexual behavior and fertilization rates depend not only on her stress levels, but also the stress level of her partner, making female sexual behavior and reproduction much more nuanced (Dickens, Balthazart et al. 2012). Rapid regulation of aromatase activity in the preoptic area of the hypothalamus of Japanese quail (Cortunix japonica) by calcium is sexually dimorphic in that the increases in aromatase activity induced by calcium are slower in females than in males, even when expression is matched between sexes (Konkle and Balthazart 2011). Studies of the effects of stress in rapidly inducing aromatase activity in Japanese quail (C. japonica) demonstrate the complexity of these mechanisms that affect sexual behavior. For instance, in males, corticosterone, arginine vasotocin (the avian homologue to arginine vasopressin), and corticotrophin-releasing factor (CRF) all played a role in increasing aromatase activity in the medial preoptic nucleus

(POM), but not in other areas of the brain expressing aromatase (Dickens,

Balthazart et al. 2012). In females, only CRF increased aromatase activity in the

POM, demonstrating the sexually dimorphic response of this brain region to various hormones (Dickens, Balthazart et al. 2012).

Other rodent species, such as prairie voles, have also been used to examine the epigenetic effects of aromatase on socio-sexual behavior. Prairie voles

(Microtus ochrogaster) are monogamous and demonstrate biparental care, but

10 pharmacological inhibition of aromatase with AI have produced mixed results

(Lonstein and De Vries 2000, Kramer, Perry et al. 2009). In some instances, exposure to AI, both prenatally and postnatally, did not alter typical parental behaviors of males or females once they reached adulthood (Lonstein and De

Vries 2000). However, female voles prenatally exposed to the same AI demonstrated higher levels of parental care compared to females treated with testosterone-propionate, while males treated with AI or flutamide (an androgen receptor blocker) showed decreased parenting behaviors (Kramer, Perry et al.

2009).

Learning and Cognition-Implications for Alzheimer’s Disease

It is well-understood that E2 affects cognition in the hippocampus, with women receiving aromatase inhibitors for breast cancer often reporting side- effects of cognitive impairments, but these effects may be species specific (Li,

Zhou et al. 2016, Gervais, Remage-Healey et al. 2019). Deficits in learning and memory are also a hallmark of Alzheimer’s disease, with women being at greater risk than men (Medway, Combarros et al. 2014).

Within the hippocampus of C57Bl/6J mice, estradiol is associated with increased dendritic spine density and is accompanied by improvements in long- term potentiation (LTP; a marker of long-term memory) and better performance on memory tests. Aromatase inhibition impairs this process in female mice but not in males (Vierk, Bayer et al. 2015). In contrast, one study including behavioral analysis of adult common marmosets (Callithrix jacchus) given daily letrozole for 4-weeks (20 µg orally), circulating estrogen decreased while E2

11 levels in the hippocampus increased in both males and females, but spatial working memory and intrinsic excitability of neurons were negatively affected, indicating that these effects of aromatase in the hippocampus may be independent of circulating E2 levels (Gervais, Remage-Healey et al. 2019).

Studies of Alzheimer’s disease (AD) also assess cognitive function and demonstrate the sexually dimorphic responses of males and females to aromatase activity in the hippocampus. Aromatase action may increase severity of

Alzheimer’s symptoms (e.g. beta-amyloid plaque formation) in male mice as demonstrated in an aromatase knockout mouse genetically crossed with an APP23 mouse (a mouse model of Alzheimer’s) to test the independent effects of testosterone on AD. Male APP23/Ar+/- mice show improved cognitive function and decreased amyloid plaque formation compared to APP23 control mice, indicating that increases in aromatase activity in the hippocampus of males may exacerbate Alzheimer’s symptoms as increases in testosterone may be protective for males (McAllister, Long et al. 2010). In humans, gene polymorphisms of

CYP19A1 are associated with performance on memory tests in middle-aged women and the role of estrogen in the development of Alzheimer’s in these groups seems complex (Butler, Warden et al. 2010). For instance, menopause is often associated with increased risk for AD but previous work has demonstrated that aromatase is upregulated in the hippocampus of postmenopausal women yet downregulated with AD, making the relationship between circulating estrogens, local aromatase activity, and their association with the development of AD complex (Kravitz, Meyer et al. 2006, Ishunina, Fischer et al. 2007, Butler,

12 Warden et al. 2010). It remains unclear exactly why these specific changes in the hippocampus of postmenopausal women occur and future research will seek to elucidate these mechanisms. Interestingly, all CYP19 gene polymorphisms identified in AD and mild cognitive impairment have been identified in women, indicating a specific role for aromatase in the development of AD in these groups

(Corbo, Gambina et al. 2009, Butler, Warden et al. 2010, Medway, Combarros et al. 2014).

This implication would likely be important for women receiving aromatase inhibitors as adjuvant treatment for postmenopausal breast cancer. It is important to note that while sex differences have not been clearly identified in humans regarding aromatase expression in the brain, abundant data demonstrates differences between men and women in various brain pathologies, such as epilepsy, schizophrenia, anxiety, and depression and aromatase may be implicated in these differences (Beyenburg, Stoffel-Wagner et al. 1999, Cherrier, Asthana et al. 2001, Butler, Warden et al. 2010, Blakemore and Naftolin 2016, Op de Macks,

Bunge et al. 2016, Alarcon, Cservenka et al. 2017).

Stress Response, Anxiety, and Depression

Mental health diagnoses (e.g. anxiety and depression) are becoming increasingly common and women are more likely to be affected than men

(Altemus, Sarvaiya et al. 2014). In addition, animal models of stress response have demonstrated sexually dimorphic patterns (Nugent, Wright et al. 2015). For instance, the gene for DNA methyltransferase (Dnmt3a) in the NAc is associated with depression/anxiety in mice and humans and contributes to susceptibility to

13 stress in males and females with responses being sexually dimorphic. Typically, male C57Bl/6J mice seem to be more resilient to subchronic variable stress than females, a pattern also seen in humans (Nugent, Wright et al. 2015).

Interestingly, overexpression of aromatase in the NAc of male mice makes them more stress resilient, whereas, deletion of Dnmt3a in the NAc makes females resilient to stress (Nugent, Wright et al. 2015).

Pharmacological aromatase inhibition also affects mood-regulating regions of the brain (e.g. amygdala) (Lynch, Dejanovic et al. 2014). For example, fear generalization is often associated with Post Traumatic Stress Disorder as it involves the generalization of fear to neutral situations (Lynch, Vanderhoof et al.

2016). The aromatization of testosterone to estradiol diminishes fear generalization in male rats, but this affect is blocked by fadrozole, an aromatase inhibitor, suggesting that E2 has anxiolytic affects in male rats (Lynch,

Vanderhoof et al. 2016). In contrast, estradiol may induce fear generalization in female rats via ERb when estradiol or an ERb agonist (DPN) is administered 24- hours prior to testing by affecting fear memory retrieval mechanisms (Lynch,

Dejanovic et al. 2014). Separately, Letrozole (2.5 mg/kg/day) for 3 weeks induces mild-anxiety in female C57BL/6J mice (Meng, Ni et al. 2011), while, in males, AI blocks the anxiolytic effects of testosterone in the dentate gyrus of male rats (Carrier, Saland et al. 2015). While the differential responses of mood- regulation in brain regions of males and females are complex, being region, sex, age, and species-specific, it is important to understand the implications of shifting hormone levels on these mental states.

14 Neuroprotection

Following traumatic brain injury or ischemic events (e.g. stroke), sex differences have been observed during recovery (Melcangi, Giatti et al. 2016). In rodents, females often fair better than males following brain injury, with estrogen and aromatase being implicated for pivotal roles in this phenomenon (Chavez,

Gogos et al. 2009, Bowen, Ferguson et al. 2011, Zhang, Wang et al. 2014,

Pietranera, Brocca et al. 2015). Current evidence supports the hypothesis that aromatase in astrocytes within the brain, as well as the general availability of E2, provides neural protection, but sex differences have not been determined (Garcia-

Segura, Wozniak et al. 1999, Melcangi, Giatti et al. 2016). Further evidence suggests that local E2 production in specific brain regions (e.g. cerebellum) is essential for neuroprotection (Lavaque, Mayen et al. 2006). Inhibition of aromatase increases neurodegeneration after administration of 3-acetylpyridine (a neurotoxin producing degeneration of inferior olivary neurons) in the cerebellum of Wistar rats, while administration of exogenous E2 decreased its effects

(Lavaque, Mayen et al. 2006). However, the molecules specifically involved in this process (aromatase, P450 side-chain cleavage (an enzyme involved in the conversion of cholesterol to pregnenolone), and StAR (regulates cholesterol transport in the mitochondria) remain unchanged in the cerebellum of female rats through the estrous cycle, suggesting that local E2 synthesis is neuroprotective independent of circulating E2 levels (Lavaque, Mayen et al. 2006, Garcia-Segura

2008).

15 Characterization of Mouse Models of Aromatase

Investigators have utilized both genetic and pharmacologic manipulations in various mammalian and non-mammalian animal models to examine the role that aromatase plays in maintaining homeostasis of steroid hormones across the lifespan with often starkly different results. In part, this is due to basic interspecies differences, as well as differences in methodological approaches (e.g. dose of E2 or aromatase inhibitors, timing of dose, or administration route of the intervention). Even within a single species, aromatase utilizes tissue-specific promoters to affect its expression (e.g. in the brain, aromatase is transcribed specifically from exon 1f of Cyp19) making translatability of results difficult

(Harada, Wakatsuki et al. 2009). The brain development of rodents resembles human fetal brain development, making rodent models suitable for examination of brain development specifically. In addition, rodent models have been essential in determining the mechanisms by which aromatase, and thus estrogen, affect the physiology of different tissues. Mouse models have been the most commonly studied mammalian model of aromatase. Transgenic mouse models with systemic, genetic aromatase ablation or tissue-specific over-expression have been created in order to more fully understand the function of the aromatase enzyme in physiology. Aromatase knockout (ArKO) mice have been developed by two different labs and have been utilized heavily in studies of estrogen’s mechanisms in physiology due to their unique lack of ability to produce estrogen in any tissue while maintaining functional estrogen receptors.

16 Two primary models of genetic aromatase ablation have been created in order to study the effects of a lack of estrogen on development and adult physiology

(Fisher, Graves et al. 1998, Honda, Harada et al. 1998). Both models present with similar phenotypes despite different genetic interruptions. Aromatase knockout mice created by the Simpson lab have a neomycin insert within exon 9 of the

Cyp19a1 gene (Fisher, Graves et al. 1998). Similarly, mice created in the Honda lab have targeted genetic disruptions of Cyp19 via neomycin inserts disrupting exons 1 and 2, as well as the brain-specific promoter region (Honda, Harada et al.

1998). The phenotype of both ArKO models present with undetectable circulating estrogen and aromatase activity, as well as increased testosterone in males and females (Fisher, Graves et al. 1998, Takeda, Toda et al. 2003, Harada,

Wakatsuki et al. 2009, Liew, Drummond et al. 2010, McAllister, Long et al.

2010). One study by Amano et al. (2017) found increased testosterone in male

ArKO mice only, while no differences were observed between WT and ArKO females, but this is the only study identified with such findings. In addition to increased endogenous testosterone, several studies characterizing the ArKO phenotype have identified increased adiposity (Honda, Harada et al. 1998,

Simpson, Clyne et al. 2002, Amano, Kondo et al. 2017), disrupted lipid metabolism (especially in males) (Simpson, Clyne et al. 2002, Hewitt, Boon et al.

2003, Hewitt, Pratis et al. 2004, Amano, Kondo et al. 2017), and reductions in fertility that differ between males and females (Honda, Harada et al. 1998,

Amano, Kondo et al. 2017).

17 With regards to obesity, ArKO mice of both sexes tend to weigh more even given a standard chow diet (Fisher, Graves et al. 1998, Simpson, Clyne et al.

2002, Takeda, Toda et al. 2003, Amano, Kondo et al. 2017). Not only is overall body weight higher, but perigonadal fat pads (considered intra-abdominal fat in humans) weigh more, indicating a greater risk for metabolic dysfunction (Fisher,

Graves et al. 1998, Simpson, Clyne et al. 2002, Amano, Kondo et al. 2017).

Jones et al. (2002) examined the increased adiposity of ArKO males and females and discovered that it is both adipocyte number and size that are increased in the

ArKO phenotype, resulting in obesity and a concurrent decrease in lean mass.

Increases in adiposity have been rescued in males and females after administration of exogenous estradiol (Simpson, Clyne et al. 2002, Hewitt, Pratis et al. 2004). The role of E2 in weight loss was further supported by Liew et al.

(2010) who saw reduced weight in ArKO males and females after administration of E2, with no effect after blockade of either androgen receptor or suppression of

GnRH secretion (Liew, Drummond et al. 2010).

Interestingly, the obesity observed in these mice by Jones et al. (2002) was not due to hyperphagia, decreased resting energy expenditure, or changes in fat oxidation, rather, it was associated with decreased physical activity, specifically in 1-year old female mice (Simpson, Clyne et al. 2002). Amano et al. (2017) observed similar behaviors with regards to food intake which did not differ from

WT mice, although ArKO mice consumed less water (Amano, Kondo et al. 2017).

In contrast, testosterone may be the primary hormone driving physical activity in male mice as aromatase inhibition had no impact on voluntary wheel running, but

18 orchidectomy reduced wheel running while supplementation with testosterone restored running to control levels (Bowen, Ferguson et al. 2011).

Lipid metabolism is also altered in the ArKO phenotype and may be sexually dimorphic (Hewitt, Boon et al. 2003). Amano et al. (2017) observed that 42-day old male ArKO mice developed hepatic steatosis, increased triglycerides (TG), total cholesterol, non-esterified fatty acids (NEFA) concentration, and phospholipid content within the liver (Amano, Kondo et al. 2017). However, female ArKO mice in this study only exhibited increases in hepatic total cholesterol concentration and phospholipid content (Amano, Kondo et al. 2017).

Providing further support for these findings, male and female ArKO mice at 1 year of age demonstrated increased cholesterol and serum HDL concentration, but serum TG concentrations were elevated only in males (Simpson, Clyne et al.

2002). Within WT mice, males have higher total cholesterol levels overall, but while these differences remain in the ArKO mouse, they are not significant

(Simpson, Clyne et al. 2002). Age may also be a factor in the development of dysregulated lipid metabolism as no differences were found in fasting serum triglycerides and total cholesterol levels of male ArKO mice at 25 weeks of age, but these measures were increased compared to WT by 36 weeks (Takeda, Toda et al. 2003). Estrogen replacement in male ArKO mice reverses the hepatic steatosis phenotype by reductions in hepatic triglyceride levels and decreases in lipid droplet size, but does not decrease hepatic cholesterol levels (Hewitt, Pratis et al. 2004).

19 Metabolic hormones such as insulin and leptin are also increased in ArKO mice, especially in males (Simpson, Clyne et al. 2002). During an IP-GTT in 18- week old ArKO males, blood glucose concentration was elevated (Takeda, Toda et al. 2003). By 24 weeks, these same males had higher fasting glucose levels and an IP injection of insulin caused a rebound increase in glucose levels 30 minutes after injection (Takeda, Toda et al. 2003). Ohlsson et al. (2017) created a mouse model that overexpresses aromatase in white adipose tissue (WAT) only (Ap2-aro mice), leaving circulating sex steroid levels unchanged (Ohlsson, Hammarstedt et al. 2017). In this model, Ap2-aro males were more insulin sensitive than WT males and expressed increased serum adiponectin, as well as upregulated Glut4 and Irs1 resulting in a model with increased insulin sensitivity and reductions in adipose tissue inflammation and enhanced adipogenesis (Ohlsson, Hammarstedt et al. 2017).

Sex hormone status and reproductive behavior and anatomy in ArKO mice are altered in males and females. While the lack of aromatase equals an inability to synthesize estrogen, estrogen receptors, androgens, and gonadotropins are also affected but can be tissue specific (Fisher, Graves et al. 1998, Liew, Drummond et al. 2010, Amano, Kondo et al. 2017). In liver, ERa mRnA levels were reduced in

ArKO females only, while androgen receptors were increased in ArKO males only (Amano, Kondo et al. 2017). Female reproductive anatomy in ArKO mice is also abnormal with ovaries and uteri being atrophied, labial folds being improperly developed, and clitoral glands being enlarged (Fisher, Graves et al.

1998, Amano, Kondo et al. 2017). Ovaries also lack corpus lutea although

20 follicles often develop normally, making ArKO females infertile (Fisher, Graves et al. 1998, Honda, Harada et al. 1998, Toda, Takeda et al. 2001, Liew,

Drummond et al. 2010). Without ovulation, progesterone remains low in ArKO females (Fisher, Graves et al. 1998). Interestingly, mitochondria in luteinized interstitial cells of ArKO mice had few lipid droplets and disorganized tubular structures when compared to WT controls, pointing to a potential mechanism for the lack of corpus lutea in ArKO females (Toda, Takeda et al. 2001). Liew et al.

(2010) discovered that it is the increase in gonadotropins, along with decreased estrogen, that is responsible for the female ArKO phenotype (Liew, Drummond et al. 2010). Interventions included E2 replacement, blockade of androgen receptor with Flutamide, or suppression of GnRH with acyline (Liew, Drummond et al.

2010). Estrogen replacement increased uterine weight, reduced the elevated LH and FSH levels in ArKO females, but had no effect on ovarian weight (Liew,

Drummond et al. 2010). Blocking AR with Flutamide had no effect on any of these variables except LH, which decreased with Flutamide treatment, but not to control levels (Liew, Drummond et al. 2010). Acyline had no effect on uterine weight, reduced FSH and LH, and decreased ovarian weight in both WT and

ArKO females (Liew, Drummond et al. 2010). Toda et al. (2001) supplemented females with E2 (15µg/day every 4 days from 4-8 weeks of age) and determined that while follicular development increased, development of the corpus luteum was not affected and therefore ovulation was not restored (Toda, Takeda et al.

2001).

21 ArKO males exhibit more impaired reproductive behaviors (e.g. mounts, intromissions, and ejaculations) while spermatogenesis is inconclusively affected.

In males, testes weights are not different between WT and ArKO and sperm remain present and active (Fisher, Graves et al. 1998, Honda, Harada et al. 1998).

However, while ArKO males from the Simpson lab sired one or more litters with heterozygous females, ArKO males from the Honda lab demonstrated impaired sexual behaviors (e.g. decreased mounting behaviors and increased latency to first mount but males sniffed and licked the female as often as WT controls. Two of

12 ArKO males were aggressive toward the female, chasing and biting her, which was not observed in the WT tests) primarily but very few litters were sired by these males, even if sexual behavior was sufficient (Fisher, Graves et al. 1998,

Honda, Harada et al. 1998). Within Simpson mice, serum LH levels were elevated while samples did not allow for testing of FSH in males (Fisher, Graves et al. 1998). These measurements were not reported in Honda mice.

Aromatase in the Nucleus Accumbens -Overview of the Brain Region

The nucleus accumbens (NAc) region is part of the mesocorticolimbic pathway in the brain and responds to stimuli such as eating, sex, drug use, and even exercise when dopamine signaling from the ventral tegmental area (VTA) projects to the NAc and prefrontal cortex (PFC) and produces a sense of

“reward”, reinforcing that particular behavior (Bonansco, Martinez-Pinto et al.

2018 2010, Tobiansky, Korol et al. 2018). Estradiol is known to be an important regulator in this pathway and may act in a sex-specific manner, with females being more affected by fluctuating hormone levels than males (Cummings,

22 Jagannathan et al. 2014). The NAc receives input primarily from dopamine and glutamate, but also expresses membrane-associated estrogen receptors alpha

(ERa) and beta (ERb), and aromatase, in addition to G protein-coupled estrogen receptor 1 (GPER1) (Cao, Dorris et al. 2016, Tonn Eisinger, Gross et al.). The

NAc consists of a core and an outer shell that are thought to have differing, but interrelated functions. The shell is thought to respond to short-term reward to a novel experience, while the NAc core seems to play a larger role in the development of patterned behaviors to obtain reward, such as in addiction

(Meredith, Baldo et al. 2008, West and Carelli 2016).

Role of Nucleus Accumbens in Motivation for Physical Activity

Numerous studies have investigated the effects of exercise on the NAc and behavioral outcomes (e.g. depression, anxiety, hedonic eating, etc.), but fewer studies have examined how changes to the NAc affect spontaneous or voluntary physical activity. Even so, it is well established that exercise induces a reward response in the NAc, but the exact mechanisms as to how this occurs are still under investigation (Park, Kanaley et al. 2016, Roberts, Ruegsegger et al. 2017,

Chen, Li et al. 2019, Zhang, He et al. 2019, Zhuang, Tan et al. 2019). For instance, previous work from our lab has shown that female rats bred for high- capacity running (HCR) not only run longer than rats bred for low-capacity running (LCR), but also exhibit increased expression of dopamine mRNA in the

NAc (Park, Kanaley et al. 2016). Another study demonstrated that genes in the

NAc of male and female rats selectively bred for high or low voluntary running behaviors were differentially expressed, but sex-comparisons were not made

23 (Padilla, Jenkins et al. 2013). It is important to note here the differences in methodologies of physical activity assessment. Spontaneous physical activity

(SPA) or locomotor activity (LA) in rodents are typically assessed in the home cage or behavioral tests (e.g. open-field test) and translates to activities in humans such as standing, fidgeting, and ambulating, whereas voluntary wheel running in rodents translates to motivated physical activity or exercise (Perez-Leighton,

Little et al. 2017).

Mu-opioid receptor, as well as orexin are both potential mechanisms by which the NAc effects spontaneous physical activity in rodents. For instance, sensitivity to orexin, a key regulator of SPA in the lateral hypothalamus, along with GABA afferents from the NAc shell contribute to differences in SPA in rats

(Thorpe and Kotz 2005, Perez-Leighton, Little et al. 2017). In addition, administration of DAMGO (a µ-opiod receptor agonist) directly into the NAc of rats increases locomotor activity in a dose-dependent manner in sedentary groups while not affecting voluntary wheel running in running groups, pointing to independent mechanisms regulating SPA and motivated physical activity (i.e. wheel running) (Lee, Parker et al. 2019).

With regards to voluntary exercise, specific strains of Wistar rats have been selectively bred for high voluntary running (HVR) or low voluntary running

(LVR) (Roberts, Ruegsegger et al. 2017). Several studies utilizing these strains have investigated dopamine receptors, protein kinase inhibitor alpha, NMDA receptors, and µ-opioid receptor in the NAc for their role in motivation for physical activity. Overexpression of protein kinase inhibitor alpha in female rat

24 NAc partially rescues low running behavior in female LVR rats while the same treatment produces no changes in WT female rats (Grigsby, Ruegsegger et al.

2019). Between adult female HVR and LVR rats, N-methyl-D-aspartate

(NMDA) receptor subtype 1 (NR1) mRNA shows greater expression in the NAc of HVR rats. In addition, there is a greater response of medium spiny neurons to administration of NMDA in NAc of female HVR rats and greater retention of dopamine content (Grigsby, Kovarik et al. 2018).

Direct manipulation of dopamine receptor or µ-opioid receptor seems to affect only HVR rats. Direct agonism or antagonism of the µ-opioid receptor within the NAc reduced nightly wheel running in HVR rats, but not LVR rats

(Ruegsegger, Toedebusch et al. 2015). A second study found that lesion of dopaminergic neurons with 6-hydroxydopamine ablated the observed decrease in running following intraperitoneal injection of naltrexone (a µ-opioid receptor antagonist) indicating the dopaminergic activity is required downstream of opioid signaling to influence voluntary activity (Ruegsegger, Brown et al. 2016). In addition, HVR rats treated with a D1 agonist or antagonist decreased nightly running behaviors while LVR rats were unaffected (Roberts, Gilpin et al. 2012).

Interestingly, cocaine elicits a greater increase in locomotor activity in LVR rats, but this response does not correlate with dopamine transporter protein in the NAc, suggesting that HVR rats are less sensitive to the rewarding properties of drugs of abuse which may highlight mechanisms in addiction (Brown, Green et al. 2015).

In terms of gene expression in the NAc of these rat strains, expression of dopamine receptor 1 (Drd1), dopamine receptor 5 (Drd5), dopamine receptor 2

25 (Drd2), nuclear receptor subfamily 4, group A, member 2 (Nr4a2), FosB, and brain-derived neurotrophic factor (Bdnf) mRNA do not differ between HVR and

LVR rats (Roberts, Gilpin et al. 2012). However, expression of genes for µ- opioid receptor and prodynorphin are higher in the NAc of adult female HVR rats

(Ruegsegger, Toedebusch et al. 2015).

Observations in WT rats and mice of both sexes have yielded similar results to those seen in HVR rats. Within female WT rats, intra-accumbens inhibition of cyclin-dependent kinase 5 (Cdk5) dose-dependently decreases wheel running (Ruegsegger, Toedebusch et al. 2017). Activation of dopamine neuron 1

(D1), as well as inhibition of D2 neurons, in the NAc of male and female

C57Bl/6J mice increased wheel running and locomotor activity. In contrast, activation of D2 neurons reduced running and locomotor activity (Zhu,

Ottenheimer et al. 2016). Within male WT mice, haloperiodol (a dopamine receptor 2 (D2) antagonist) reduced voluntary wheel running, but this effect was attenuated with genetic adenosine receptor deletion in the NAc core as adenosine receptor expression is normally co-localized with D2 receptor (Correa, Pardo et al. 2016). Overall, it is clear that the mechanisms driving motivation for physical activity are complex and most likely involve multiple pathways. Future studies of these behaviors will need to further elucidate these mechanisms.

Effects of Estrogen Loss on Physical Activity

It is well-known that removal of estrogen via menopause or surgical ovariectomy produces weight gain and metabolic dysfunction, specifically in women, and E2 supplementation reverses these changes (Ley, Lees et al. 1992, Svendsen,

26 Hassager et al. 1995, Stefanska, Bergmann et al. 2015, Clookey, Welly et al.

2019). Reductions in physical activity also often occurs with reductions in estrogen signaling, compounding the risks for metabolic dysfunction (Booth,

Roberts et al. 2012, Duval, Prud'homme et al. 2013). Despite this evidence, aromatase inhibitors (AI) are often used as adjuvant hormone therapy in postmenopausal women with breast cancer, further reducing local E2 synthesis and promoting further metabolic issues (Brown, Mao et al. 2014, de Paulo, Viezel et al. 2017). In one study assessing these effects, healthy women demonstrated an inverse relationship between physical activity levels and lipid profile, fasting glucose, and c-reactive protein as expected, while this relationship did not exist in women receiving AI for breast cancer, indicating that increased physical activity does not coincide with improved metabolic function in these women (de Paulo,

Viezel et al. 2017). Notably, reductions in physical activity in women receiving

AI are often associated with arthralgia and musculoskeletal symptoms (e.g. lower limb joint pain or reduced physical function) (Brown, Mao et al. 2014). Another study assessing quality of life in women receiving various hormonal treatments for breast cancer (e.g. tamoxifen or AI) showed that exercise may improve quality of life for women taking aromatase inhibitors (Park, Tish Knobf et al. 2019).

In a separate exercise intervention study, postmenopausal women receiving AI for breast cancer reported ~30-40% of the recommended 150 mins of aerobic exercise per week prior to the start of the exercise intervention.

Fortunately, a 12-month exercise intervention (supervised resistance training

2x/week (whole-body, 3 sets, 8-12 reps @ max weight to complete 12 repetitions;

27 150 mins of moderate-intensity aerobic exercise/week) improved body composition, however, it did not change bone mineral density, contrary to expectations as resistance training has been shown to improve bone mineral density (Thomas, Cartmel et al. 2017). While this study demonstrates that exercise improves body composition in these women, inclusion in the study required low participation in physical activity for the year prior to recruitment.

This limits the ability to extrapolate the influence of AI on motivation for physical activity.

Numerous studies in animals have exhibited similar results to those in humans. Ovariectomy has repeatedly been shown to reduce physical activity, especially in females (Jones, Thorburn et al. 2000, Izumo, Ishibashi et al. 2012,

Vieira-Potter, Padilla et al. 2015, Park, Kanaley et al. 2016). Wild-type female rodents are consistently more active than males when assessed in various manners

(e.g. open-field test, voluntary wheel running) and gonadectomy reduces some of these differences in both sexes. In females, E2 supplementation restores activity in ovariectomized females, but has no effect in castrated males (Careau, Bininda-

Emonds et al. 2012, Rosenfeld 2017). Furthermore, Cyp19a1 expression was not affected in the NAc of male Long-Evans rats 6 weeks after gonadectomy, but analysis of physical activity was not performed (Tobiansky, Korol et al. 2018).

Future studies should aim to determine the connection between changing estrogen levels, both locally and in the blood, and motivation for physical activity as well as the mechanisms by which this occurs.

28 Known Role of Estrogen in the Nucleus Accumbens

Estrogens role in the NAc has been studied primarily with regards to addiction, although physical activity produces the same neurochemical reward as drugs of abuse (Bonansco, Martinez-Pinto et al. 2018 2010, Tobiansky, Korol et al. 2018). Females are more susceptible to the rewarding properties of drugs of abuse and addiction than males, potentially due to estrogen’s actions in the NAc

(Peterson, Mermelstein et al. 2015, Satta, Certa et al. 2018). This susceptibility to addiction is attenuated with the removal of ovarian hormones and restored with the administration of estradiol (Peterson, Mermelstein et al. 2015). Specifically, in female mice, estrogen via ERb increases conditioned place preference for cocaine, indicating that females prefer cocaine-laced water over normal water when estrogen is present in the NAc (Satta, Certa et al. 2018). Metabotropic glutamate receptor-5 (mGluR5) is also a potential mechanism (specifically in female rats and Syrian hamsters) by which estrogen works to decrease dendritic spine density in the NAc core, an attribute known to be associated with addiction

(Staffend, Loftus et al. 2011, Peterson, Mermelstein et al. 2015). In addition, E2 potentially modulates dopamine transport by increasing its availability via inhibition of dopamine reuptake in the striatum and NAc of rats (Almey, Milner et al. 2015).

ArKO mouse models have been used to assess susceptibility to addiction in the absence of E2. Female ArKO mice show reduced locomotor response to stimulant drugs (e.g. amphetamine) compared to WT females, while male ArKO mice also exhibit a diminished response, but to a lesser extent. Dopamine

29 transporters were significantly higher in the caudate putamen of male ArKO mice compared to WT, while this increase was not seen in females, suggesting a compensatory mechanism for dopaminergic regulation in male mice (Chavez,

Gogos et al. 2009).

In contrast to the above findings, similar results are observed between sexes in adult WT rats after direct inactivation of the NAc. When a behavior, such as wheel running, is habitualized, rats will demonstrate recovery or increased running behavior after a period of forced abstinence from the wheel. This response is greater in adult WT female rats compared to WT males, but inactivation of the NAc via direct administration of Bupivacaine or muscimol decrease this rebound response in both sexes (Basso and Morrell 2015). These findings suggest that the nucleus accumbens core (which is associated with habitualized behavioral reinforcement) may be differentially affected between sexes when compared to the NAc shell (which is associated with reward for novel behaviors) (Meredith, Baldo et al. 2008, West and Carelli 2016).

Conclusions

Aromatase is responsible for local E2 synthesis in multiple tissues and is highly conserved across species (Naftolin, Ryan et al. 1972, Naftolin, Ryan et al.

1975, Simpson, Zhao et al. 1997, Simpson 2000, Ohlsson, Hammarstedt et al.

2017). Within the brain, aromatase is present in many regions and exerts its effects on behavior via varying mechanisms (Beyenburg, Stoffel-Wagner et al.

1999, Cherrier, Asthana et al. 2001, Simpson, Clyne et al. 2002, Murray, Hien et al. 2009, Butler, Warden et al. 2010, Kurian, Olesen et al. 2010, Schwarz, Nugent

30 et al. 2010, Blakemore and Naftolin 2016, Op de Macks, Bunge et al. 2016,

Alarcon, Cservenka et al. 2017, Patel 2017, Rosenfeld 2017). The nucleus accumbens is a brain region of particular interest with regards to motivation for physical activity, as it is this region that is implicated in the mechanisms associated with pleasure and reward, in addition to addiction (Park, Kanaley et al.

2016, Roberts, Ruegsegger et al. 2017, Bonansco, Martinez-Pinto et al. 2018

2010, Tobiansky, Korol et al. 2018, Chen, Li et al. 2019, Zhang, He et al. 2019,

Zhuang, Tan et al. 2019). Female rodents, as well as humans, are more susceptible to fluctuations in both circulating E2 levels, as well as local E2 levels due to aromatase, by responding with greater changes in behavior when compared to males who do not always exhibit phenotypic changes with alterations in E2 or aromatase (Chavez, Gogos et al. 2009, Corbo, Gambina et al. 2009, Butler,

Warden et al. 2010, Bowen, Ferguson et al. 2011, Altemus, Sarvaiya et al. 2014,

Medway, Combarros et al. 2014, Zhang, Wang et al. 2014, Peterson, Mermelstein et al. 2015, Pietranera, Brocca et al. 2015, Satta, Certa et al. 2018). The same observation holds true in females regarding assessments of physical activity and dopamine signaling in animals and humans, and so shifts in E2 in the NAc may affect motivation for exercise, specifically in females, via an estrogenic- dopaminergic mechanism in this brain region (Jones, Thorburn et al. 2000, Booth,

Roberts et al. 2012, Duval, Prud'homme et al. 2013, Peterson, Mermelstein et al.

2015). In males, one study in particular observed that males may possess a compensatory mechanism for dopaminergic regulation that protects them from

31 some of the behavioral changes observed in females (Chavez, Gogos et al. 2009).

Future studies should seek to examine this further.

Study Rationale and Specific Aims

Previous work assessing the phenotype of ArKO mice has not always made comparisons between sex for all variables tested. More often, either males, or females were selected for an entire study or only a single sex was tested for specific behaviors while the other sex remained unknown with regards to that variable (e.g. levels of spontaneous physical activity). In addition, RNAseq methods of evaluating gene expression in the brain regions associated with these behaviors, specifically in the NAc, have not been previously performed to our knowledge. Given this work in assessing the phenotype of aromatase knockout mice, we sought to make direct sex comparisons in WT and aromatase knockout

(ArKO) mice of metabolic phenotype and behaviors (e.g. sleep, learning, spontaneous physical activity). The aim of this study is to assess to 12-16-week- old male and female WT and ArKO mice for bodyweight, body composition

(EchoMRI), sleep (MET chambers), spatial memory (Barnes maze), spontaneous physical activity (MET chambers), and anxiety-like behavior (Elevated Plus

Maze). In addition, RNAseq analyses will be performed to assess differences in gene expression in the NAc brain region of all groups. Pair-wise comparisons of gene expression will be made between groups and correlated to phenotypic behaviors. We hypothesize that ArKO mice will be less active than WT counterparts and that gene expression in the NAc will correlate with these differences in physical activity.

32 Chapter 2-Effect of aromatase ablation on phenotype and NAc gene expression in male and female mice. 1. Introduction Declining estrogen concentrations during menopause, as well as suppression of estrogen synthesis due to genetic or pharmacological means, can result in metabolic dysfunction, including insulin resistance and increased visceral adiposity. This has been observed in rodent models and humans (Gibb, Homer et al. 2016, Park, Kanaley et al. 2016, Muller, Keiler et al. 2018). Notably, females experience an associated decrease in physical activity with reductions in circulating estrogen, yet these effects have not been observed to the same extent with estrogen suppression in males (Bowen, Ferguson et al. 2011, Park, Kanaley et al. 2016, Muller, Keiler et al. 2018). Mechanisms for the decrease in voluntary physical activity in women have not been fully elucidated. Importantly, exercise has been shown to improve estrogen-induced metabolic disruptions (Bergeron,

Mentor et al. 2014, MacDonald, Ritchie et al. 2015, Gorres-Martens, Field et al.

2018).

Aromatase is required to convert testosterone to estrogen in both sexes across vertebrate species (Blakemore and Naftolin 2016). The highest concentrations are in the gonads, but this enzyme is also present in the brain and adipose tissue, where it acts in a paracrine manner (Nelson and Bulun 2001).

Female rodents with high amounts of circulating estrogen are more active than males (Park, Kanaley et al. 2016, Bartling, Al-Robaiy et al. 2017, Klinker,

Kohnemann et al. 2017), yet genetic ablation of the aromatase enzyme reduces locomotor activity more dramatically than ovariectomy alone (Jones, Thorburn et

33 al. 2000). This finding suggests that aromatase activity in the brain is necessary to maintain a certain level of physical activity, even when ovarian estrogen production ceases.

Previous work from our laboratory has shown that the decrease in physical activity that occurs after ovariectomy correlates with suppression of dopamine- associated gene expression in the nucleus accumbens (NAc) (Park, Kanaley et al.

2016). This brain region is part of the mesolimbic pathway that plays a prominent role in the reward circuitry of the brain, relying on dopamine signaling to learn rewarding behaviors (e.g. food, sex, drugs of abuse [e.g. cocaine and amphetamine]) and increases motivation to repeat those behaviors (Gorres-

Martens, Field et al. 2018, Tonn Eisinger, Larson et al. 2018). Estrogen increases susceptibility of females to addiction by modulating the density of dopamine receptors in the NAc (Becker, Perry et al. 2012) and exercise yields analogous effects (Mathes, Nehrenberg et al. 2010, Rosenfeld 2017). Collectively, these results suggest that estrogen signaling in the NAc may modulate dopamine signaling, affecting motivation for physical activity, at least in females (Park,

Kanaley et al. 2016). These findings in rodents point to potential implications of the manipulation of aromatase activity and its physiological effects in humans.

Aromatase inhibition (AI) is commonly used as adjuvant therapy for post- menopausal women with hormone-receptor positive breast cancer and the incidence of cognitive impairments increases with such treatments (Jenkins,

Shilling et al. 2004, Collins, Mackenzie et al. 2009). Together with the metabolic dysfunction that already occurs post menopause, AI therapy may exacerbate these

34 issues by decreasing motivation for engaging in physical activity. In addition, males are also prescribed aromatase inhibitors, often to treat the decrease in endogenous testosterone that occurs with increasing age, but the cognitive effects of AI in men have not been thoroughly assessed (Dias, Melvin et al. 2016). Thus, it is important to determine whether there are sex differences in risk for physical inactivity due to aromatase suppression and if so, the underlying molecular changes that might be driving such effects.

We hypothesized that physical activity would be significantly depressed in aromatase deficient females but not aromatase deficient males. Additionally, it was postulated that NAc gene expression patterns would be differentially regulated by sex and correlate with the degree of physical inactivity. To test these hypotheses, a whole-body aromatase knockout (ArKO) mouse model created by

Ogawa et al (Honda, Harada et al. 1998) was utilized to examine for sex and genotype differences in spontaneous physical activity, as well as other behaviors

(e.g., anxiety-like behaviors). In addition, RNA-seq was performed on the NAc brain regions of both sexes and both genotypes in order to examine for potential sex- and genotype-dependent associations between gene expression disruptions in the NAc and overall physical activity.

2. Materials and Methods

2.1 Subjects

Male and female C57Bl6/J wild-type (WT) and aromatase knockout (ArKO) mice were bred and housed at University of Missouri Animal Sciences Research

Center (Columbia, MO, USA) under a normal 12-12 h light-dark cycle with room

35 temperature maintained at 21.7°C (71°F) with food (standard chow, Purina 5008) and water available ad libitum. All mice were weaned at 21 days of age and housed singly or in pairs based on sex and genotype. At 12 weeks of age, all mice were divided based on sex (M/F) and genotype (WT/KO) into the following groups: male

(WT, n=8; KO, n=10) and female (WT, n=11; KO, n=9). Pair-housing was utilized to provide social enrichment, as recommended by our ACUC committee.

Some mice were singly housed, if necessary, to avoid aggressive behaviors depending on age and sex-matching. All animal husbandry and experimental procedures were conducted in accordance with the NIH Guide for Care and Use of

Laboratory Animals and approved by the University of Missouri Institutional

Animal Care and Use Committee (ACUC, Protocol # 8573) prior to initiation of experiments.

2.2 Metabolic Chambers

At 12 weeks of age, animals were placed in the indirect calorimetry (MET) chambers (Promethion; Sable Systems International, Las Vegas, Nevada) to assess metabolic activity parameters including total energy expenditure (TEE), resting energy expenditure (REE), and spontaneous physical activity (SPA) by the summation of x-, y-, and z-axis beam breaks. Body weight and food intake were measured before and after each 72-hour (h) period in the MET chambers. Data from the first 24-h period was not used as this time was considered the habituation period. The 48-h run captured at least two light and two dark cycles of REE.

2.3 Body Composition

Percent body fat (BF%) and percent lean mass were measured by a nuclear

36 magnetic resonance imaging whole-body composition analyzer (EchoMRI

4in1/1100; Echo Medical Systems, Houston, TX).

2.4 Energy Intake

At 13 weeks of age, all mice were given a premeasured amount of food

(standard chow, Purina 5008). Mice were allowed to eat ad libitum for 5 days in their home cage and left undisturbed except for daily checks to ensure food and water remained in the cage. On the fifth day, the remaining amount of food was measured and subtracted from the premeasured food weight to determine the total weight of food in grams that was consumed by that cage over the five days. The total was averaged per day, as well as per mouse if pair-housed.

2.5 Barnes Maze

At 14 weeks of age, Barnes maze testing was performed to assess spatial memory and learning. The maze was constructed with an opaque white plexiglass platform with twelve escape holes around the perimeter of the floor of the maze.

All holes were spaced approximately 30 degrees apart with 4 brightly colored geometric shapes placed at 90º intervals along the maze wall which served as visual cues to guide the mouse toward the proper escape hole. During each trial, eleven of the twelve holes remained closed, while the twelfth hole remained open to an escape box into which the mouse could run. As a slightly aversive, non- painful motivation to enter the escape hole, a bright light was placed over the maze (1200 lx v. testing room light 400 lx). Each mouse was randomly assigned a hole in the maze which remained consistent over the 5-day test period. Two trials were run each day, with a 30 min rest period between trials, for 5 consecutive

37 days following the Barnes maze protocol as described previously with the exceptions detailed below (Rosenfeld and Ferguson 2014). Briefly, mice were acclimated to the test environment for 30 mins prior to the start of the first trial each day and each mouse was placed in the center of the maze under a clean polypropylene box. At the beginning of each trial, the box was removed, and a timer was started. Each mouse was given 300s to explore the maze and find the correct hole. At the end of each trial, the mouse was shown the correct hole by gently placing the mouse into the escape box. Once each trial was finished, the maze, escape hole box, and polypropylene cover box were cleaned with a 70% ethanol solution.

Each trial was recorded using a Sony HD Handycam HDR-CX440 (San

Diego, CA) camcorder and videos were analyzed using the ANY-Maze (Stoetling

Co., Wood Dale, IL, USA) software v. 6.0 analysis system. Indices measured include total distance traveled (m), total time mobile (s), total time immobile (s), number of entries to the correct hole zone, and latency to first entry to the correct hole zone (s).

It should be noted that this maze is designed for the mice to escape from the lights by dropping or jumping into the box under the assigned escape hole.

Surprisingly, after the first week of testing, none of the mice had escaped into the box. To compensate for this behavior, the analysis focuses on assessing the mouse’s behavior during the entire 300 s trial.

2.6 Elevated Plus Maze (EPM)

The EPM is used to examine exploratory and anxiety-like behaviors in

38 mice and was conducted as described previously (Fountain, Mao et al. 2008,

Jasarevic, Sieli et al. 2011). Briefly, each animal was placed in the center of the maze and was allowed to explore for a single 300 s trial. Each trial was recorded with a Sony HD Handycam HDR-CX440 (San Diego, CA) camcorder and videos were analyzed using Observer 11 software (Noldus Technologies, Leesburg, VA,

USA). Indices measured included number of entries into the center of the maze, duration of time spent in the center (s), number of entries into the open arms of the maze, time spent in the open arms (s), number of rearing instances, duration of head dipping behavior below the level of the platform (s), and duration of time spent mobile, all of which are considered to be exploratory, non-anxious behaviors. Behaviors examined that are considered more anxiety-like are number of entries and time spent (s) in the closed arms of the maze, number and duration

(s) of grooming events, and time spent immobile.

2.7 Brain Collection & Preparation

After animals were humanely euthanized in accordance with AVMA guidelines for euthanasia of animals, whole brain was collected and snap-frozen in liquid nitrogen and stored at -80°C until transcriptome analyses. Whole brains were rapidly thawed at room temperature to obtain tissue punches of the NAc region. This brain region was located and dissected using the Allen mouse brain atlas (Lein, Hawrylycz et al. 2007). Brain slices were taken using a Zivic brain block (Adult mouse brain slicer matrix with 1.0 mm coronal section slice intervals; Zivic Instruments, Pittsburgh, PA) as a guide. Micro punch samples were taken of the nucleus accumbens using a 2 mm Harris micro punch (GE

39 Whatman Harris micro punch, 2mm; GE Healthcare Bio-sciences, Marlborough,

MA). Bilateral NAc punches were combined and snap-frozen in liquid nitrogen and stored at -80°C until further analyses.

2.8 RNA isolation of NAc region

NAc samples were homogenized in Qiazol solution using a tissue homogenizer (TissueLyser LT, Qiagen, Valencia, CA). Total RNA was isolated according to the Qiagen’s RNeasy lipid tissue protocol and assayed using a

Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE) to assess purity and concentration. The five highest concentrations within each group in the young age-range were submitted to the University of Missouri DNA Core facility for RNA-seq analyses. A Qubit fluorometer (Invitrogen, Carlsbad, CA) with the Qubit HS RNA assay kit was used to verify sample concentration. RNA integrity was assessed using the Fragment Analyzer (Advanced Analytical

Technologies, Ankey, IA) automated electrophoresis system. Only those samples that had a RIN score above 7.0 were used for follow-up RNA-seq analysis.

2.9 RNA-seq

High-throughput sequencing was performed at the University of Missouri

DNA Core Facility (Columbia, MO) as described previously (Ortega, Bivens et al. 2019). Libraries were constructed following the manufacturer’s protocol with reagents supplied in Illimina’s TruSeq mRNA Stranded Library Preparation kit.

Briefly, the poly-A containing mRNA is purified from total RNA. RNA is fragmented and double-stranded cDNA is generated from fragmented RNA, and the index containing adapters are ligated to the ends. The final construct of each

40 purified library was evaluated using the Fragment Analyzer automated electrophoresis system, quantified with the Qubit flourometer using the Qubit HS dsDNA assay kit, and diluted according to Illumina’s standard sequencing protocol for sequencing on the NextSeq 500.

2.10 Analysis of RNA-seq data

RNA-seq data was processed and analyzed as described previously

(Givan, Bottoms et al. 2012). Briefly, latent Illumina adapter sequence were identified and removed from input 100-mer RNA-Seq data using Cutadapt

(Martin 2011). Subsequently, input RNA-Seq reads were trimmed and filtered to remove low quality nucleotide calls and whole reads, respectively, using the

Fastx-Toolkit (GitHub). To generate the final set of quality-controlled RNA-Seq reads, foreign or undesirable sequences were removed by similarity matching to the Phi-X genome (Genome Resource) (NC_001422.1), the relevant ribosomal

RNA genes as downloaded from the National Center for Biotechnology

Information (Information-NCBI) or repeat elements in RepBase (Jurka,

Kapitonov et al. 2005), using Bowtie (Langmead, Trapnell et al. 2009). This final set of quality-controlled RNA-Seq reads was aligned to the Ensemble Mus musculus genome sequence, GRCm38.p5 (Ensembl), using STAR (Dobin, Davis et al. 2013) with the default settings, which also generates the initial expression estimates for each annotated gene. The R (Foundation) Bioconductor

(Bioconductor 2003) package DESeq2 (Love, Huber et al.) is used to normalize the gene expression estimates across the samples and to analyze the differential expression of genes between sample types. A gene is identified as being

41 differentially expressed between two conditions when the FDR-corrected p-value of its expression ratio is less than 0.05. Subsequent data was reformatted, sorted and filtered using a variety of R commands (Foundation) and Bash command-line scripts (System), which are available upon request.

In order to further understand the role of the identified DEGs, gene ontology (GO) terms were analyzed for molecular function (MF) and biological process (BP), in addition to KEGG and Reactome biological pathways using gprofiler (Raudvere, Kolberg et al.). Benjamini-Hochberg false discovery rate

(FDR) threshold was set at 0.02. Protein-protein interaction (PPI) of the DEGs was analyzed using the Search Tool for the Retrieval of Interacting Genes

(STRING database v.11.0) (Szklarczyk, Gable et al.).

2.11 qPCR analysis of NAc

Following RNA extraction from the NAc (as described above), total RNA was assayed using a Nanodrop spectrophotometer (Thermo Scientific, Wilmington,

DE) to assess purity and concentration. First-strand cDNA was synthesized from total RNA using the High Capacity cDNA Reverse Transcription kit (Applied

Biosystems, Carlsbad, CA). Quantitative real-time PCR was performed as previously described using the ABI StepOne Plus sequence detection system

(Applied Biosystems) (Roseguini, Mehmet Soylu et al. 2010, Padilla, Jenkins et al. 2013). Primer sequences were designed using the NCBI Primer Design tool.

All primers were purchased from Sigma-Aldrich (St. Louis, MO) or Integrated

DNA Technologies (Carolville, IA). The internal house-keeping control gene

(HKG) used was 18s and cycle threshold (CT) was not different among the

42 groups of animals. mRNA expression was calculated by 2∆CT where ∆CT = 18s

CT - gene of interest CT and presented as fold-difference. mRNA levels were normalized to the F WT group which was set at 1.

2.12 Statistics

Between groups, 2 x 2 ANOVA were performed to determine main effects of genotype (G), sex (S), and G x S interactions on all metabolic and behavioral data. When an interaction occurred, Tukey’s post-hoc analyses were performed to determine independent group differences. All data are presented as mean ±

SEM; n=8-11/group and p values < 0.05 were considered statistically significant.

Effect sizes were calculated using partial eta-squared for ANOVAs. Analyses were performed using SPSS version 25 (IBM, Armonk, New York).

Pearson correlations were calculated for the 7 differentially expressed genes identified in female mice and considered significant if p < 0.05. A strong correlation was defined as having an R-value > ± 0.7, a moderate correlation having an R-value of ± 0.5-0.7, and a weak correlation having an R-value < ± 0.5

(Mukaka 2012).

43 3. Results For all variables examined, sex and/or genotype differences were reported.

Male (M) wild-type (WT; n=8) and aromatase knockout (ArKO; n=10) were compared to female (F) WT (n=11) and ArKO (n=9) mice. Significant sex-by- genotype (SxG) interactions were noted, as well as main effects of both sex (S) and genotype (G).

3.1 Body weight, fat mass, lean mass, and body composition

2 A SxG interaction (F(3, 34) = 47.791, p < 0.01, � = 0.584) was observed for total body weight at time of sacrifice, such that female WT mice weighed less than male WT mice, whereas females (but not males) were affected by aromatase gene ablation. Specifically, female ArKO gained more weight compared to female

WT, whereas males did not differ by genotype. This resulted in the female WT weighing less than all other groups (p < 0.05) (Fig. 3A). SxG interactions were

2 also observed for total fat mass (F(3, 34) = 9.472, p < 0.01, � = 0.218) and total

2 lean mass (F(3,34) = 50.431, p < 0.01, � = 0.597). Here, females were more adversely affected by aromatase ablation in terms of fat mass, such that they gained more fat (but not lean) mass. Remarkably, this resulted in female ArKO mice having significantly more fat than all other groups (Fig. 3B). In terms of lean mass, similar to total body weight, female WT had lower lean mass than male WT, while females gained, and males reduced lean mass with aromatase ablation (Fig. 3C). These differential changes in fat and lean mass resulted in the

ArKO mice of both sexes having significantly higher body fat percentages than

2 respective WT mice (main effect of genotype, F(3,34) = 18.787, p < 0.01, � =

2 0.356) (Fig. 3D). Similarly, main effects of both sex (F(3,34) = 11.786, p < 0.01, �

44 2 = 0.257) and genotype (F(3,34) = 19.321, p < 0.01, � = 0.362) were observed for lean mass percentage such that females and ArKOs had lower percent lean mass

(Fig. 3E).

3.2 Energy Intake & Expenditure, Respiratory Quotient, and Physical Activity

A SxG interaction was observed for energy intake when expressed relative

2 to total body weight (F(3,33) = 12.724, p < 0.01, � = 0.278) such that WT females consumed more relative energy than WT males, yet only females are affected by aromatase ablation. That is, females reduced relative energy intake whereas males were not affected by aromatase ablation. This resulted in WT females having significantly higher energy intake than all other groups (p < 0.01) (Fig. 4A).

When total energy expenditure was expressed relative to total body

2 weight, a SxG interaction was observed for the light (F(3,34) = 24.458, p < 0.01, �

45 2 = 0.419), dark (F(3,34) = 18.115, p < 0.01, � = 0.348), and 24 hr. (F(3,34) = 21.094, p < 0.01, �2 = 0.383) cycles. For all three time periods, WT females demonstrated significantly higher energy expenditure than all other groups (p <

0.01) (Fig. 4B). No significant differences were observed for respiratory quotient

(Fig. 4C) whereas WT females had greater resting energy expenditure than all other groups (SxG, F (3,34) = 23.134, p < 0.01, �2 = 0.405) (Fig. 4D).

Spontaneous physical activity, expressed as “ped meters” (i.e., total meters traveled in cage), revealed a SxG interaction for both the light (F(3,34) = 10.134, p

2 2 < 0.01, � = 0.230) and dark cycles (F(3,34) = 15.956, p < 0.01, � = 0.319), as well

2 as the 24 hour activity (F(3,34) = 16.116, p < 0.01, � = 0.322). Post-hoc analyses revealed that, for each time period, WT females were significantly more active than all other groups (p < 0.05) (Fig. 4E). In addition, ArKO females were less active than WT males with a trending significance during the dark cycle and 24- hour total (SPA-Dk: p = 0.079; SPA-Total: p = 0.095).

46

3.3 Sleep, Anxiety-Like Behavior, and Learning

Genotype, as well as sex, affected sleep duration such that a SxG interaction occurred during each cycle (p < 0.05). During the light (i.e., rodent

2 inactive) cycle (F(3,34) = 7.464, p < 0.05, � = 0.180), male and female WT did not differ in sleep quantity, while aromatase deletion affected females (but not males) such that ArKO females slept more than WT females (p < 0.01) but did not differ from either male group (Fig. 5A). During the dark (i.e., rodent active) cycle

2 (F(3,34) = 18.955, p < 0.01, � = 0.332), WT males slept significantly more than females whereas only females were affected by aromatase deletion. That is, female ArKO slept more in the active cycle than WT females, resulting in this group sleeping more than all other groups (p < 0.05). Similarly, over the 24 hr

2 period (F(3,34) = 15.147, p < 0.01, � = 0.332), WT females slept less than all other groups (p < 0.01).

47 The elevated plus maze (EPM) was used to assess anxiety-like behavior.

A main effect of genotype was observed for the duration of time spent in the

2 center of the maze (F(3,33) = 2.428, p < .05, � = .174) such that ArKO mice spent less time in the center compared to WT mice, but no differences were observed between groups for the duration in the closed and open arms of the maze (Fig.

5B). In addition, a SxG interaction was observed for duration mobile and

2 immobile in the EPM (F(3,33) = 2.424, p < .05, � = .133), such that male ArKO mice increased time spent mobile, while female ArKO mice decreased time spent mobile although there were no significant post hoc differences between groups (p

= .08) (Fig. 5C). No differences were observed regarding the number of entries into each zone of the EPM, as well as the frequency or duration of grooming events, rearing, or head dips while in the maze (data not shown). Together, these findings suggest that aromatase deletion does not affect anxiety-like behavior, yet the differences in locomotor behavior during the EPM align with the previous findings regarding the sex-specific effects of aromatase deletion on physical activity behavior.

Barnes Maze testing was utilized to assess short-term memory and learning in all groups. No significant differences were observed for duration mobile or immobile in the Barnes maze (Fig. 5D), total distance travelled (Fig.

5E), or number of entries to the correct hole zone (data not shown). Together, these findings indicate that aromatase ablation does not affect memory and learning in either sex, at least in young mice.

48

3.4 Analysis of Gene Expression

In order to investigate possible mechanisms for the decrease in physical activity in female ArKO mice, RNASeq data were collected in the nucleus accumbens (NAc) brain region for male and female WT and ArKO mice.

49

3.4.1 Sex Differences in NAc Gene Expression Profiles

The top 30 differentially expressed genes (DEGs) were identified in females and expressed as a fold change from their male counterpart (Fig. 6a).

Gene descriptions are explained in Table 3 (Stelzer, Rosen et al. 2016, UniProt

2019). Of these 30 DEGs, six (Xist, Ddx3y, Kdm5d, Eif2s3y, Uty, and Shox2) showed a fold-change (FC) > 2. The majority of DEGs identified were downregulated in females compared to males. Only 8 DEGs were upregulated, including Ddx3y, Kdm5d, Eif2s3y, Uty, Per3, Scara3, Vcl, and Tmprss6.

50 Wild-type (WT) mice were used to identify normal sex differences in NAc gene expression. In WT mice, 25 genes were differentially expressed in females compared to males and presented here. Of those, the top DEGs are associated with X-chromosome inactivation (Xist, Jpx), ATP binding (Ddx3y), histone demethylation (Uty,Kdm6a, Kdm5c) , protein synthesis (Eif2s3y, Eif2s3x), estrogen receptor (Esr1), regulator of CDK kinases (Ccnd1; misregulated in cancer), Prostaglandin E3 (PGE3) receptor (Ptger3), regulation of circadian rhythm (Per3), development of the actin cytoskeleton (Rnd1, Vcl), response to oxidative stress (Scara3), organ/skeletal development and healing (Pdgfra), beta- adrenergic signaling (Ngfr), neutrophil degranulation (Ddx3x, Plekho2), DNA repair (Nabp1) and growth regulation (Shox2).

Of the 25 differentially expressed genes in WT male vs. female, the following genes were not different between sexes in the ArKO (i.e., these sex differences were due to differences in aromatase expression): Pbdc1, Esr1,

Ccnd1, Tmem176a, Ptger3, Per3, Rnd1, Scara3, Pdgfra, Vcl, Tmprss6, Ngfr,

Plekho2, Nabp1, and Shox2.(Fig. 6a). Of these unique DEGs in WT mice, the following genes were downregulated in females compared to males: Pbdc1, Esr1,

Ccnd1, Tmem176a, Ptger3, Rnd1, Pdgfra, Ngfr, Plekho2, Nabp1, and Shox2 (Fig.

6a)

In ArKO mice, 5 DEGs were uniquely downregulated in the knockout mouse, including: Mid1, Scrg1, P2ry1, Rpl38, and Dchs2 (Fig. 6a). These genes are associated with formation of microtubules in the cytoplasm (Mid1), neurodegenerative changes in prion disease (Scrg1), extracellular receptors for

51 ATP/ADP (P2ry1), ribosomal components (Rpl38), and cell adhesion that may be involved in Alzheimer’s disease (Dchs2) (Fig. 6a).

52 Table 3: Gene descriptions of top 30 DEGs between male and female WT & ArKO mice.

Gene name, protein name, description, fold change in female from male counterpart, and FDR of the genes expressed in Fig. 6a.

3.4.2 Genotype Differences in NAc Transcriptome Profiles

The top 30 differentially expressed genes were identified in ArKO mice when compared to their WT counterpart (Fig. 6b). Gene descriptions are

53 explained in Table 4 (Stelzer, Rosen et al. 2016, UniProt 2019). Of these 30

DEGs, only 1 gene (Gldn) shows a FC > 2 in females and males had 2 genes

(Cyp19a1 & Bhlhe22) that exhibited a FC > 2 in the knockout.

Overall, the 30 most significant DEGs based on p-value are presented between WT and ArKO mice (Fig. 6b). Within males, 15 genes were upregulated in the ArKO compared to the WT and are associated with regulation of cell determination/differentiation (Bhlhe22), conversion of pyruvate to acetyl-CoA

(Dlat), neuron migration, growth, and survival, as well as cell adhesion (Ndnf), motion sickness (Tanc1), nucleic acid binding (Zmat4), stress response (Hspa8,

Stk39, Dnaja4), regulation of neuronal positioning in the brain (Dab1), anticoagulant synthesis (Hs3st1), lipid binding and metabolic pathways (Plekha2), negative regulation of transcription factors (Id2), reversible hydration of carbon dioxide (Car4), mediation of neuropeptide Y, a GI hormone involved in satiety

(Npy1r), and formation of extracellular matrix glycoproteins (Efemp1) (Fig. 6b).

Cyp19a1, the gene code for the aromatase enzyme, was paradoxically upregulated in the ArKO male. Further analyses are being performed in order to validate the gene knockout. It is hypothesized that RNAseq data used primers for an undisrupted exon of the Cyp19 gene, while our mice are disrupted within exons 1 and 2, potentially creating a feedback to upregulate aromatase due to the absence of estrogen.

The following DEGs were downregulated in the ArKO male compared to

WT and are associated with: BH4 synthesis (Pts), clustering of sodium channels within the Nodes of Ranvier in the peripheral nervous system (Gldn), stress

54 response (Cryab), glycosylation of amino acids (Nxpe4), vitamin A synthesis

(Bco2), calcium-activated chloride channels involved in olfaction (Ano2), formation of alpha-ketoglutarate receptor 1 (Oxgr1), microtubules in the cytoplasm (Mid1), exocytosis of vesicles necessary for olfactory signaling

(Syt10), platelet aggregation (P2ry1), and spermatogenesis (Spata13) (Fig. 6b).

Twenty three of the 30 DEGs expressed in Figure 4b were unique to the male. The following DEGs were also present in female mice: Pts, Gldn, Cryab,

Cyp19a1, and Nxpe4 (Fig. 6b). There are only 2 DEGs that are unique to the female mouse. Both are downregulated and are associated with binding of carbohydrate and hyaluronic acid (Layn) and positive regulation of the Wnt signaling pathway (Dixdc1) (Fig. 6b). Cyp19a1 was also paradoxically upregulated in the female ArKO, as in the male, and further tests are being run in order to validate the model.

55

56 Table 4: Gene descriptions of top 30 DEGs between WT & ArKO male and female mice

Gene name, protein name, description, fold change in female from male counterpart, and FDR of the genes expressed in Fig. 6b.

57 3.5 Correlation of gene expression to phenotypical traits and behaviors

The top 6 DEGs identified between WT and ArKO female mice (Pts,

Layn, Dixdc1, Per3, Cryab, Nxpe4, Gldn) were chosen for correlation analysis with selected phenotypical traits across all mice (Fig. 5a). The same correlation analyses were repeated in female mice only (Fig. 5b).

3.5.1 Correlations Across All Cohorts of Mice

Across all mice, a strong negative correlation was observed between

Cryab (a member of the small heat shock protein family that prevents protein aggregation under stress) and sleep during the dark cycle (r = -0.70, p < 0.01). In addition, Cryab correlates positively with energy intake relative to body weight (r

= 0.85, p < 0.01) (Fig. 7a). Gldn (required for proper clustering of sodium channels on Nodes of Ranvier in the peripheral nervous system) correlated positively with energy intake relative to body weight (r = 0.82, p < 0.01). Per3 (a

CLOCK gene that regulates circadian rhythm) correlated strongly in a positive direction with both lean mass (r = 0.81, p < 0.01) and total body weight (r = 0.79, p < 0.01) but exhibited a strong negative correlation with relative energy intake (r

= -0.83, p < 0.01) (Fig. 7a). One particular gene of interest, Pts (necessary for

BH4 synthesis, and thus production of catecholamines), exhibited only a moderate negative correlation with body fat (r = -0.55, p < 0.05) and a moderate positive correlation with energy intake relative to body weight (r = 0.55, p < 0.05), but did not correlate strongly with any behavior analyzed.

58

59 3.5.2 Correlations in female mice only

When assessing correlations in female mice only, numerous strong correlations were observed (Fig. 7b). Thirteen of the 18 phenotypic traits examined correlated strongly with one or more of the 7 DEGs chosen, with physical activity being of particular interest. Per3 correlated strongly in a negative direction with physical activity during all cycles (rLt =-0.72, pLt < 0.05; rDk = -0.81, pDk < 0.01; r24 = -0.81, p24 < 0.01) , as well as relative energy intake (r

= -0.85, p < 0.01) and correlated positively with sleep during the dark cycle (r =

0.66, p < 0.05), when mice are typically active (Fig. 7b). Pts, Layn (gene for a protein involved in binding of carbohydrate and hyaluronic acid), Dixdc1 (a positive regulator of the Wnt pathway), Cryab, and Gldn also correlated positively with relative energy intake (r = 0.90, p < 0.01; r = 0.83, p < 0.01; r =

0.85, p < 0.01; r = 0.93, p < 0.01; r = 0.92, p < 0.01 respectively) (Fig. 7b).

Interestingly, Pts, Layn, and Dixdc1 correlated strongly with all phases of physical activity in a positive direction (rLt = 0.73, 0.78, & 0.70 respectively, p <

0.05 for all; rDk = 0.90, 0.83, & 0.87 respectively, p < 0.01 for all; r24 = 0.88, 0.86,

& 0.85 respectively, p < 0.01 for all) but correlated negatively with sleep during the dark cycle (r = -0.87, -0.83, -0.75 respectively, p < 0.05 for all) (Fig. 7b).

Cryab, and Gldn also correlated negatively with sleep during the dark cycle (r = -

0.78 & -0.73 respectively, p < 0.05 for all), while Layn and Cryab correlated negatively with sleep during the light cycle (r = -0.70 & -0.74 respectively, p <

0.05 for all) (Fig. 7b). Nxpe4 (gene for a protein associated with glycosylation of amino acids) positively correlated with physical activity during the dark (r = 0.85,

60 p < 0.01) and 24 hr cycles (r = 0.76, p < 0.05), while Gldn exhibited a strong positive correlation with physical activity during the light cycle only (r = 0.70, p <

0.05) (Fig. 7b).

61

62

3.6 Pathway enrichment and protein-protein interaction of DEGs

A protein-protein interaction (PPI) network of DEGs was built on the basis of STRING analysis results for pairwise comparisons between both sex and genotype (Fig. 8A-D). In addition, pathway enrichment was evaluated by gene ontology (GO) of DEGs for molecular function (MF), biological processes (BP), and KEGG and Reactome biological pathways. PPI analyses were performed in order to identify the most likely interactions between the observed DEGs and any potential downstream proteins of interest. Gene ontology was analyzed in order to determine which biological pathways may be affected by differentially expressed genes based on sex or genotype.

63

3.6.1 Analysis of DEGs function in WT males v. females

Gene ontology (GO) analysis of DEGs in WT mice, male v. female, indicated involvement in molecular function (MF) of histone demethylation activity, carbohydrate derivative binding, steroid and nuclear hormone receptor binding, estrogen response element binding, and estrogen receptor activity (Table

5). The top 27 biological processes (BP) associated with DEGs between male and female WT mice involving steroid hormones, metabolism, or other relevant physiological processes associated with aromatase (e.g. sleep/circadian rhythm,

64 DNA methylation, etc.) were selected and expressed in Table 5. Three biological pathways (Reactome) were identified as HDMs demethylase histones, chromatin organization, and RUNX1 regulation of estrogen receptor mediated transcription

(Table 5). All KEGG biological pathways identified were associated with cancer, endocrine resistance, or thyroid hormone signaling pathway (Table 5). PPI network of the top 25 DEGs in WT male and female mice are presented in Fig.

8B.

65

3.6.2 Analysis of DEGs function in ArKO males v. females

GO analysis of DEGs in ArKO mice, male v. female, indicated involvement in MF of histone demethylase activity, carbohydrate derivative binding, steroid hormone receptor binding, Type 1 metabotropic glutamate

66 receptor binding, estrogen response element binding, nuclear hormone receptor binding, estrogen receptor activity, and hormone receptor binding (Table 6). The top 29 BP associated with DEGs between male and female ArKO mice involving steroid hormones, metabolism, or other relevant physiological processes associated with aromatase (e.g. sexual differentiation, growth and development, ovulation, etc.) were selected and expressed in Table 6. Ten biological pathways

(Reactome) were identified as: HDMs demethylate histones; chromatin modifying enzymes and organization; negative regulation of the PI3K/AKT network; NFG and proNGF binds to p75NTR; PI5P, PP2A and IER3 Regulate PI3K/AKT signaling; signal transduction; RUNX1 regulation of estrogen receptor mediated transcription; neutrophil degranulation; and axonal growth stimulation (Table 6).

KEGG biological pathways identified were associated with cancer, endocrine resistance, or thyroid hormone signaling pathway, focal adhesion, human cytomegalovirus infection, PI3K-Akt signaling, prolactin signaling, JAK-STAT signaling, calcium signaling, RNA transport, RAP1 signaling, regulation of the actin cytoskeleton, viral carcinogenesis, and the RAS signaling pathway (Table

6). PPI network of the top 16 DEGs in ArKO male and female mice are presented in Fig. 8A.

67

68 3.6.3 Analysis of DEGs function in Male WT v. ArKO In addition to sex differences, genotype comparisons were also made within sex. GO analysis of DEGs in male mice, WT v. ArKO, indicated involvement in MF of lipid binding, prostaglandin binding, 6- pyruvoyltetrahydropterin synthase (Pts) activity, carbohydrate derivative binding, and both neuropeptide Y receptor and peptide YY receptor activity (Table 7).

The top 32 biological processes (BP) associated with DEGs between WT and

ArKO male mice involving steroid hormones, metabolism, or other relevant physiological processes associated with aromatase (e.g. growth and development of the CNS, locomotor behavior, aging, etc.) were selected and expressed in

Table 7. Four biological pathways (Reactome) were identified as: Class A/1 rhodopsin-like receptors, metabolism, Ga signaling events, and GPCR ligand binding (Table 7). Only one KEGG biological pathway was identified as a longevity regulating pathway (Table 7). PPI network of the top 28 DEGs in WT and ArKO male mice are presented in Fig. 8D.

69

3.6.4 Analysis of DEGs and PPI in Females WT v. ArKO GO analysis of DEGs in female mice, WT v. ArKO, indicated involvement in MF of 6-pyruvoyltetrahydropterin synthase (Pts) activity, aromatase activity, and axon function (Table 8). No BP were identified as associated with DEGs between WT and ArKO female mice. Seven biological

70 pathways (Reactome) were identified as: BH4 synthesis, recycling, salvage, and regulation; metabolism of cofactors; estrogen biosynthesis; HSF1-dependent transactivation; endogenous sterols; and metabolism of steroid hormones (Table

8). Five KEGG biological pathways were identified as folate biosynthesis, steroid hormone biosynthesis, ovarian steroidogenesis, metabolic pathways, and longevity regulating pathway-multiple species (Table 8). PPI network of the top

7 DEGs in WT and ArKO female mice are presented in Fig. 8C.

4. Discussion In this study, WT and ArKO mice were analyzed in order to determine phenotypic traits that are differentially expressed between sex and genotype. In addition, novel RNAseq data was acquired in the NAc brain region and correlated to metabolic variables. Finally, gene ontology pathway enrichment and protein- protein interaction analyses were performed to assess the most commonly associated genes differentially expressed from our genes of interest, as well as the biological pathways most likely to be different between sex and genotype.

71 Characterization of the aromatase knockout phenotype in mice has previously established that ArKO mice tend to be obese even provided a standard chow diet (Fisher, Graves et al. 1998, Honda, Harada et al. 1998, Jones, Thorburn et al.), with impaired lipid metabolism (Jones, Thorburn et al. 2000, Nemoto,

Toda et al. , Hewitt, Boon et al. 2003, Hewitt, Pratis et al. 2004, Amano, Kondo et al. 2017), disrupted sexual behavior (Honda, Harada et al. 1998, Dalla, Antoniou et al. 2004), and reduced locomotor activity (Jones, Thorburn et al. 2000).

Gonadotropin assays consistently show elevated testosterone levels in male mice, but mixed results in females (Fisher, Graves et al. 1998, Toda, Takeda et al. 2001,

Takeda, Toda et al. 2003, Harada, Wakatsuki et al. 2009, Liew, Drummond et al.

2010, Amano, Kondo et al. 2017).

In our current study, sex differences, as well as genotypic effects support previous findings in relation to increased obesity and reduced physical activity in

ArKO mice, especially females. The current work though builds on these original findings by screening for gene expression differences in the NAc region, that may account for differences in physical activity and ensuing obesity.

Our findings demonstrate that within WT mice, sex differences exist such that females tend to weigh less than males, with less lean mass and similar body fat percentages. However, when aromatase is absent in females, body weight increases similar to WT males. In addition, ArKO females have increased fat mass beyond all other groups. These findings suggest that this is due to reduced physical activity, rather than increases in energy intake or decreased resting energy expenditure. In contrast, ArKO males did not significantly change body

72 weight or fat mass from WT males. Surprisingly, ArKO males had decreased lean mass from WT counterparts. Testosterone tends to increase lean mass

(Mouser, Loprinzi et al.). The current study did not examine steroid hormone concentrations in these mice, but previous work suggests that endogenous testosterone is increased in ArKO males (Amano, Kondo et al. 2017).

Further supporting our findings in females, ArKO females sleep more during the dark cycle when mice are most typically active, directly relating to their decreases in physical activity, yet males did not differ between genotype.

Other variables, such as anxiety-like and learning behaviors did not significantly change with loss of aromatase in males or females. This is in contrast to previous findings. Martin et al. (2003) demonstrated impaired short-term spatial reference memory in male and female ArKO mice using a Y-maze (Martin, Jones et al.

2003). The use of a Y-maze rather than a Barnes maze may explain the differences in findings, but, similar findings have been observed in male and female ArKO in assessments of anxiety-like behavior in the EPM (Dalla,

Antoniou et al. 2004, Dalla, Antoniou et al. 2005).

Sex differences in NAc gene expression as determined by RNAseq were novel findings in this study. Within WT mice, sex differences indicated that upregulated genes in WT females were associated with ATP binding, RNA binding, and the formation of intramolecular interactions, lysine demethylation, initiation of protein synthesis, and histone demethylation. These upregulations were shared with similar sex differences in ArKO males and females.

73 Two specific genes upregulated in WT and ArKO females (Ddx3y and

Kdm5d) are associated with male infertility, while mutations in the male-specific histone demethylase Uty are associated with Kabuki syndrome, a rare autosomal dominant disorder characterized by skeletal abnormalities, short stature, heart defects, intellectual disability, and obesity. Interestingly, Uty is also upregulated in WT and ArKO females. Four of the 15 DEGs uniquely expressed in WT mice are upregulated in females and associated with regulation of circadian rhythm

(Per3), depletion of reactive oxygen species (Scara3), actin filament protein binding (Vcl), and potentially matrix remodeling in the liver (Tmprss6).

Down regulated genes in WT and ArKO females (compared to male counterparts) are associated with x-chromosome inactivation (Xist and JPX), neutrophil degranulation (Ddx3x), the early steps of protein synthesis (Eif2s3x), lysine-specific demethylation (Kdm6a and Kdm5c). Mutations within these genes are associated with rare congenital disorders such as: Kabuki Syndrome, congenital mental retardation, and Mehmo Syndrome, an x-linked intellectual disability characterized by epilepsy, mental retardation, microcephaly, hypogenitalism and obesity. In addition to these, ArKO females exhibit 5 DEGs unique to their genotype. These genes are all downregulated in the ArKO female compared to the ArKO male and are associated with microtubule formation, neurodegenerative changes in prion disease, receptors for extracellular ATP/ADP, ribosomal components, and cell adhesion potentially implicated in Alzheimer’s disease, compressive strength, and appendicular lean mass.

74 When comparing genotypes within sex, 170 DEGs were identified in males while only 7 were identified in females. The 30 most significant DEGs in males were presented in this paper. In males, the upregulated genes in the ArKO male from WT are involved in glycolysis, neuronal function, vascular endothelial function, nucleic acid binding, and stress response. Downregulated genes unique in the ArKO male compared to WT are associated with vitamin A synthesis, heat shock protein binding, cell signaling, inflammation, and microtubule formation.

Interestingly, of the 7 DEGs in females, only 2 of these DEGs are unique to the ArKO, and both are downregulated from WT. These include Layn and

Dixdc1 which serve to influence the binding of carbohydrate and hyaluronic acid and as a positive regulator of the Wnt pathway respectively. This finding points to a potential mechanism for the role of aromatase in the expression of Dixdc1 and subsequently neuronal patterning and organogenesis during embryonic development (Komiya and Habas 2008), as well as carcinogenesis in adults

(Zhan, Rindtorff et al. 2017).

The remaining 5 DEGs in female mice are involved in BH4 synthesis, heat shock proteins, sodium channel function on Nodes of Ranvier in the peripheral nervous system, and glycosolation of amino acids. Our hypothesis that NAc gene expression would correlate with decreases in physical activity in female mice was supported in genes Pts, Layn, Dixdc1, and Gldn during the light cycle. Pts, Layn, and Dixdc1 also correlated with SPA during the dark cycle, but Gldn did not correlate as strongly during this phase. Nxpe4, which did not correlate with SPA during the light cycle, strongly correlated with SPA during the dark cycle and

75 maintained that correlation in the 24 hr total. Not surprisingly, these same genes negatively correlated with sleep during all phases. These correlations only hold true however in females. When all mice are analyzed for the same gene correlations with SPA and sleep, no significance was found. Supporting these findings, a previous study genetically downregulated systemic Pts expression in

C57Bl/6J mice resulting in lowered BH4 synthesis in liver and brain, but unaffected brain monoamines (Korner, Scherer et al. 2016). Their findings determined that downregulation of Pts contributed to increased body weight and elevated intrabdominal fat, particularly in males with alterations in glucose and lipid metabolism in both sexes potentially via reduced eNOS function (Korner,

Scherer et al. 2016). Analyses of behaviors that contribute to obesity (e.g. reduced physical activity) were not analyzed.

We were initially surprised to see that Cyp19a1 was increased in male and female ArKO mice since this gene should be dysfunctional in our model. We hypothesized that this is due to specific strain differences in this mouse model.

Our ArKO mice are disrupted at exons 1 and 2 of Cyp19a1, while other strains of

ArKO are disrupted at exon 9 (Fisher, Graves et al. 1998, Honda, Harada et al.

1998). It is possible that our RNAseq data used primers specific for exon 9 of

Cyp19a1 and that there is a negative feedback leading to increases in the non- functioning aromatase gene. Further analyses are being performed in order to test this hypothesis utilizing both Western blot for aromatase protein expression, as well as q-pcr with primers for exons 1 and 2 of Cyp19a1.

76 Paradoxically, pathway enrichment and functional analysis identified locomotion as a GO:BP pathway between male WT and ArKO mice, but no significant differences were observed in our study regarding male physical activity. Previous work has identified dopamine in the NAc as differentially regulated with changes in physical activity (Park, Kanaley et al. 2016, Bonansco,

Martinez-Pinto et al. 2018, Jardi, Laurent et al. 2018). This brain region may be sexually dimorphic in that females reduce physical activity after loss of estrogen, while males rely on testosterone for the same behaviors, an idea which is supported by studies of addiction and locomotor activity, behaviors that are also associated with the NAc and have been shown to be sexually dimorphic

(Wissman, McCollum et al. 2011, Zhu, Ottenheimer et al. 2016, Jardi, Laurent et al. 2018). Future studies need to investigate these mechanisms further.

In conclusion, our findings of ArKO phenotype sex differences support previous work in this model. Novel findings in NAc gene expression and their correlations with physical activity, sleep, and body composition provide insight to possible mechanisms of the role of estrogen via aromatase. Future studies will focus on examining the underpinning mechanisms leading to sex differences in physical activity that may be regulated by the NAc.

77 Chapter 3- Study Limitations and Future Directions

Limitations of this study include the use of a genetic, systemic aromatase knockout mouse model. While aromatase is present in the brain of all mammalian species, the translatability of these findings to humans is limited. Future studies in humans could utilize fluorescent imaging techniques to analyze aromatase expression and activity in vivo, allowing scientists to investigate which brain regions are most affected by changes in aromatase.

In addition, because these mice lack the aromatase enzyme throughout fetal development, it is not possible to determine if the observed effects of aromatase are organizational or activational in nature. Future studies in WT mice may examine aromatase action in the NAc by pharmacologic manipulation via localized cranial injection of aromatase inhibitors or use of Designer Receptors

Exclusively Activated by Designer Drugs (DREADD) technology targeting dopaminergic neurons in order to assess the organizational versus activational role of aromatase in this brain region. In addition, metabolic and behavioral analyses would be performed in order to evaluate the physiologic effects of the changes in aromatase and dopamine signaling on these outcomes. These findings will have strong implications for clinical treatments of various diseases associated with E2 signaling, in particular, the metabolic dysfunction and insulin resistance associated with obesity.

78 Further limitations include the Barnes maze protocol. Since none of the mice would voluntarily escape into the hole of the maze, protocol had to be shifted to accommodate these behaviors. Final results of our alternative protocol were still relevant, although they were limited in nature. A different maze design may be more suited for C57Bl/6J mice strains.

Also, motivation was not directly tested in this experiment as we did not anticipate collection of the RNAseq data when the project began. The DNA Core

Facility grant that allowed for this analysis was received after experiments had begun, so we did not change the study design in order to remain consistent across all groups. The use of a conditioned place preference behavioral test, along with assessments of voluntary wheel running, may be more suited when determining the role of aromatase in the NAc regarding motivation for physical activity.

79 5. References

(2016). "Committee Opinion No. 663: Aromatase Inhibitors in Gynecologic Practice." Obstet Gynecol 127(6): e170-174.

Abdelgadir, S. E., J. A. Resko, S. R. Ojeda, E. D. Lephart, M. J. McPhaul and C. E. Roselli (1994). "Androgens regulate aromatase cytochrome P450 messenger ribonucleic acid in rat brain." Endocrinology 135(1): 395-401.

Alarcon, G., A. Cservenka and B. J. Nagel (2017). "Adolescent neural response to reward is related to participant sex and task motivation." Brain Cogn 111: 51-62.

Almey, A., T. A. Milner and W. G. Brake (2015). "Estrogen receptors in the central nervous system and their implication for dopamine-dependent cognition in females." Horm Behav 74: 125-138.

Altemus, M., N. Sarvaiya and C. Neill Epperson (2014). "Sex differences in anxiety and depression clinical perspectives." Front Neuroendocrinol 35(3): 320- 330.

Amano, A., Y. Kondo, Y. Noda, M. Ohta, N. Kawanishi, S. Machida, K. Mitsuhashi, T. Senmaru, M. Fukui, O. Takaoka, T. Mori, J. Kitawaki, M. Ono, T. Saibara, H. Obayashi and A. Ishigami (2017). "Abnormal lipid/lipoprotein metabolism and high plasma testosterone levels in male but not female aromatase- knockout mice." Arch Biochem Biophys 622: 47-58.

Bakker, J., C. De Mees, Q. Douhard, J. Balthazart, P. Gabant, J. Szpirer and C. Szpirer (2006). "Alpha-fetoprotein protects the developing female mouse brain from masculinization and defeminization by estrogens." Nat Neurosci 9(2): 220- 226.

Bartling, B., S. Al-Robaiy, H. Lehnich, L. Binder, B. Hiebl and A. Simm (2017). "Sex-related differences in the wheel-running activity of mice decline with increasing age." Exp Gerontol 87(Pt B): 139-147.

Basso, J. C. and J. I. Morrell (2015). "The medial prefrontal cortex and nucleus accumbens mediate the motivation for voluntary wheel running in the rat." Behav Neurosci 129(4): 457-472.

Becker, J. B., A. N. Perry and C. Westenbroek (2012). "Sex differences in the neural mechanisms mediating addiction: a new synthesis and hypothesis." Biol Sex Differ 3(1): 14.

Bergeron, R., J. S. Mentor, I. Cote, E. T. Ngo Sock, R. Rabasa-Lhoret and J. M. Lavoie (2014). "Loss of ovarian estrogens causes only mild deterioration of

80 glucose homeostasis in female ZDF rats preventable by voluntary running exercise." Horm Metab Res 46(11): 774-781.

Beyenburg, S., B. Stoffel-Wagner, M. Watzka, I. Blumcke, J. Bauer, J. Schramm, F. Bidlingmaier and C. E. Elger (1999). "Expression of cytochrome P450scc mRNA in the hippocampus of patients with temporal lobe epilepsy." Neuroreport 10(14): 3067-3070.

Biegon, A., S. W. Kim, D. L. Alexoff, M. Jayne, P. Carter, B. Hubbard, P. King, J. Logan, L. Muench, D. Pareto, D. Schlyer, C. Shea, F. Telang, G. J. Wang, Y. Xu and J. S. Fowler (2010). "Unique distribution of aromatase in the human brain: in vivo studies with PET and [N-methyl-11C]vorozole." Synapse 64(11): 801-807.

Bioconductor. (2003). "Bioconductor." Retrieved 12th October 2016, from http://bioconductor.org/.

Blakemore, J. and F. Naftolin (2016). "Aromatase: Contributions to Physiology and Disease in Women and Men." Physiology (Bethesda) 31(4): 258-269.

Bonansco, C., J. Martinez-Pinto, R. A. Silva, V. B. Velasquez, A. Martorell, M. V. Selva, P. Espinosa, P. R. Moya, G. Cruz, M. E. Andres and R. Sotomayor- Zarate (2018). "Neonatal exposure to estradiol increases dopaminergic transmission in Nucleus Accumbens and morphine-induced conditioned place preference in adult female rats." J Neuroendocrinol.

Boon, W. C., J. D. Chow and E. R. Simpson (2010). "The multiple roles of estrogens and the enzyme aromatase." Prog Brain Res 181: 209-232.

Booth, F. W., C. K. Roberts and M. J. Laye (2012). "Lack of exercise is a major cause of chronic diseases." Compr Physiol 2(2): 1143-1211.

Bowen, R. S., D. P. Ferguson and J. T. Lightfoot (2011). "Effects of Aromatase Inhibition on the Physical Activity Levels of Male Mice." J Steroids Horm Sci 1(1): 1-7.

Brown, J. C., J. J. Mao, C. Stricker, W. T. Hwang, K. S. Tan and K. H. Schmitz (2014). "Aromatase inhibitor associated musculoskeletal symptoms are associated with reduced physical activity among breast cancer survivors." Breast J 20(1): 22- 28.

Brown, J. D., C. L. Green, I. M. Arthur, F. W. Booth and D. K. Miller (2015). "Cocaine-induced locomotor activity in rats selectively bred for low and high voluntary running behavior." Psychopharmacology (Berl) 232(4): 673-681.

81 Bulun, S. E. (2014). "Aromatase and estrogen receptor alpha deficiency." Fertil Steril 101(2): 323-329.

Butler, H. T., D. R. Warden, E. Hogervorst, J. Ragoussis, A. D. Smith and D. J. Lehmann (2010). "Association of the aromatase gene with Alzheimer's disease in women." Neurosci Lett 468(3): 202-206.

Cao, J., D. M. Dorris and J. Meitzen (2016). "Neonatal Masculinization Blocks Increased Excitatory Synaptic Input in Female Rat Nucleus Accumbens Core." Endocrinology 157(8): 3181-3196.

Cao, M., J. Duan, N. Cheng, X. Zhong, Z. Wang, W. Hu and H. Zhao (2012). "Sexually dimorphic and ontogenetic expression of dmrt1, cyp19a1a and cyp19a1b in Gobiocypris rarus." Comp Biochem Physiol A Mol Integr Physiol 162(4): 303-309.

Careau, V., O. R. Bininda-Emonds, G. Ordonez and T. Garland, Jr. (2012). "Are voluntary wheel running and open-field behavior correlated in mice? Different answers from comparative and artificial selection approaches." Behav Genet 42(5): 830-844.

Carrier, N., S. K. Saland, F. Duclot, H. He, R. Mercer and M. Kabbaj (2015). "The Anxiolytic and Antidepressant-like Effects of Testosterone and Estrogen in Gonadectomized Male Rats." Biol Psychiatry 78(4): 259-269.

Chavez, C., A. Gogos, M. E. Jones and M. van den Buuse (2009). "Psychotropic drug-induced locomotor hyperactivity and prepulse inhibition regulation in male and female aromatase knockout (ArKO) mice: role of dopamine D1 and D2 receptors and dopamine transporters." Psychopharmacology (Berl) 206(2): 267- 279.

Chen, W., J. Li, J. Liu, D. Wang and L. Hou (2019). "Aerobic Exercise Improves Food Reward Systems in Obese Rats via Insulin Signaling Regulation of Dopamine Levels in the Nucleus Accumbens." ACS Chem Neurosci.

Cherrier, M. M., S. Asthana, S. Plymate, L. Baker, A. M. Matsumoto, E. Peskind, M. A. Raskind, K. Brodkin, W. Bremner, A. Petrova, S. LaTendresse and S. Craft (2001). "Testosterone supplementation improves spatial and verbal memory in healthy older men." Neurology 57(1): 80-88.

Clookey, S. L., R. J. Welly, D. Shay, M. L. Woodford, K. L. Fritsche, R. S. Rector, J. Padilla, D. B. Lubahn and V. J. Vieira-Potter (2019). "Beta 3 Adrenergic Receptor Activation Rescues Metabolic Dysfunction in Female Estrogen Receptor Alpha-Null Mice." Front Physiol 10: 9.

82 Collins, B., J. Mackenzie, A. Stewart, C. Bielajew and S. Verma (2009). "Cognitive effects of hormonal therapy in early stage breast cancer patients: a prospective study." Psychooncology 18(8): 811-821.

Corbo, R. M., G. Gambina, L. Ulizzi, G. Moretto and R. Scacchi (2009). "Genetic variation of CYP19 (aromatase) gene influences age at onset of Alzheimer's disease in women." Dement Geriatr Cogn Disord 27(6): 513-518.

Correa, M., M. Pardo, P. Bayarri, L. Lopez-Cruz, N. San Miguel, O. Valverde, C. Ledent and J. D. Salamone (2016). "Choosing voluntary exercise over sucrose consumption depends upon dopamine transmission: effects of haloperidol in wild type and adenosine A(2)AKO mice." Psychopharmacology (Berl) 233(3): 393- 404.

Cummings, J. A., L. Jagannathan, L. R. Jackson and J. B. Becker (2014). "Sex differences in the effects of estradiol in the nucleus accumbens and striatum on the response to cocaine: neurochemistry and behavior." Drug Alcohol Depend 135: 22-28.

Dalla, C., K. Antoniou, Z. Papadopoulou-Daifoti, J. Balthazart and J. Bakker (2004). "Oestrogen-deficient female aromatase knockout (ArKO) mice exhibit depressive-like symptomatology." Eur J Neurosci 20(1): 217-228.

Dalla, C., K. Antoniou, Z. Papadopoulou-Daifoti, J. Balthazart and J. Bakker (2005). "Male aromatase-knockout mice exhibit normal levels of activity, anxiety and "depressive-like" symptomatology." Behav Brain Res 163(2): 186-193.

De Mees, C., J. F. Laes, J. Bakker, J. Smitz, B. Hennuy, P. Van Vooren, P. Gabant, J. Szpirer and C. Szpirer (2006). "Alpha-fetoprotein controls female fertility and prenatal development of the gonadotropin-releasing hormone pathway through an antiestrogenic action." Mol Cell Biol 26(5): 2012-2018. de Paulo, T. R. S., J. Viezel, B. L. Aro, S. C. Seidinger, A. Trindade, D. G. D. Christofaro and I. F. J. Freitas (2017). "Relationship between physical activity practice and metabolic profile of postmenopausal women under treatment with aromatase inhibitors for breast cancer." Eur J Obstet Gynecol Reprod Biol 216: 33-37.

Dias, J. P., D. Melvin, E. M. Simonsick, O. Carlson, M. D. Shardell, L. Ferrucci, C. W. Chia, S. Basaria and J. M. Egan (2016). "Effects of aromatase inhibition vs. testosterone in older men with low testosterone: randomized-controlled trial." Andrology 4(1): 33-40.

Dickens, M. J., J. Balthazart and C. A. Cornil (2012). "Brain aromatase and circulating corticosterone are rapidly regulated by combined acute stress and

83 sexual interaction in a sex-specific manner." J Neuroendocrinol 24(10): 1322- 1334.

Dobin, A., C. A. Davis, F. Schlesinger, J. Drenkow, C. Zaleski, S. Jha, P. Batut, M. Chaisson and T. R. Gingeras (2013). "STAR: ultrafast universal RNA-seq aligner." Bioinformatics 29(1): 15-21.

Duval, K., D. Prud'homme, R. Rabasa-Lhoret, I. Strychar, M. Brochu, J. M. Lavoie and E. Doucet (2013). "Effects of the menopausal transition on energy expenditure: a MONET Group Study." Eur J Clin Nutr 67(4): 407-411.

Ensembl. (2019). "Mus musculus - Ensembl Release 90." Retrieved 27th September 2017, from http://www.ensembl.org/Mus_musculus/Info/Index.

Fisher, C. R., K. H. Graves, A. F. Parlow and E. R. Simpson (1998). "Characterization of mice deficient in aromatase (ArKO) because of targeted disruption of the cyp19 gene." Proc Natl Acad Sci U S A 95(12): 6965-6970.

Foundation, T. R. "R: The R Project for Statistical Computing." from https://www.r-project.org/.

Fountain, E. D., J. Mao, J. J. Whyte, K. E. Mueller, M. R. Ellersieck, M. J. Will, R. M. Roberts, R. Macdonald and C. S. Rosenfeld (2008). "Effects of diets enriched in omega-3 and omega-6 polyunsaturated fatty acids on offspring sex- ratio and maternal behavior in mice." Biol Reprod 78(2): 211-217.

Garcia-Segura, L. M. (2008). "Aromatase in the brain: not just for reproduction anymore." J Neuroendocrinol 20(6): 705-712.

Garcia-Segura, L. M., A. Wozniak, I. Azcoitia, J. R. Rodriguez, R. E. Hutchison and J. B. Hutchison (1999). "Aromatase expression by astrocytes after brain injury: implications for local estrogen formation in brain repair." Neuroscience 89(2): 567-578. Genome Resource, N. (1993, 07/06/2018). "Enterobacteria phage phiX174 sensu lato (ID 4241)." Retrieved 22nd April 2016, from http://www.ncbi.nlm.nih.gov/genome/?term=NC_001422.1.

Gentry, A. L., K. I. Erickson, S. M. Sereika, F. E. Casillo, M. E. Crisafio, P. T. Donahue, G. A. Grove, A. L. Marsland, J. C. Watt and C. M. Bender (2018). "Protocol for Exercise Program in Cancer and Cognition (EPICC): A randomized controlled trial of the effects of aerobic exercise on cognitive function in postmenopausal women with breast cancer receiving aromatase inhibitor therapy." Contemp Clin Trials 67: 109-115.

Gervais, N. J., L. Remage-Healey, J. R. Starrett, D. J. Pollak, J. A. Mong and A.

84 Lacreuse (2019). "Adverse Effects of Aromatase Inhibition on the Brain and Behavior in a Nonhuman Primate." J Neurosci 39(5): 918-928.

Gibb, F. W., N. Z. Homer, A. M. Faqehi, R. Upreti, D. E. Livingstone, K. J. McInnes, R. Andrew and B. R. Walker (2016). "Aromatase Inhibition Reduces Insulin Sensitivity in Healthy Men." J Clin Endocrinol Metab 101(5): 2040-2046.

GitHub. (2010). "FASTX toolkit." Retrieved 30th April 2015, from https://github.com/agordon/fastx_toolkit.

Givan, S. A., C. A. Bottoms and W. G. Spollen (2012). "Computational analysis of RNA-seq." Methods Mol Biol 883: 201-219.

Gorres-Martens, B. K., T. J. Field, E. R. Schmidt and K. A. Munger (2018). "Exercise prevents HFD- and OVX-induced type 2 diabetes risk factors by decreasing fat storage and improving fuel utilization." Physiol Rep 6(13): e13783.

Grigsby, K. B., C. M. Kovarik, G. E. Rottinghaus and F. W. Booth (2018). "High and low nightly running behavior associates with nucleus accumbens N-Methyl- d-aspartate receptor (NMDAR) NR1 subunit expression and NMDAR functional differences." Neurosci Lett 671: 50-55.

Grigsby, K. B., G. N. Ruegsegger, T. E. Childs and F. W. Booth (2019). "Overexpression of Protein Kinase Inhibitor Alpha Reverses Rat Low Voluntary Running Behavior." Mol Neurobiol 56(3): 1782-1797.

Halper, A., B. Sanchez, J. S. Hodges, D. R. Dengel, A. Petryk and K. Sarafoglou (2019). "Use of an aromatase inhibitor in children with congenital adrenal hyperplasia: Impact of anastrozole on bone mineral density and visceral adipose tissue." Clin Endocrinol (Oxf). Harada, N., T. Wakatsuki, N. Aste, N. Yoshimura and S. I. Honda (2009). "Functional analysis of neurosteroidal oestrogen using gene-disrupted and transgenic mice." J Neuroendocrinol 21(4): 365-369.

Hero, M., S. Maury, E. Luotoniemi, E. Service and L. Dunkel (2010). "Cognitive effects of aromatase inhibitor therapy in peripubertal boys." Eur J Endocrinol 163(1): 149-155.

Hero, M., E. Norjavaara and L. Dunkel (2005). "Inhibition of estrogen biosynthesis with a potent aromatase inhibitor increases predicted adult height in boys with idiopathic short stature: a randomized controlled trial." J Clin Endocrinol Metab 90(12): 6396-6402.

Hewitt, K. N., W. C. Boon, Y. Murata, M. E. Jones and E. R. Simpson (2003). "The aromatase knockout mouse presents with a sexually dimorphic disruption to cholesterol homeostasis." Endocrinology 144(9): 3895-3903.

85

Hewitt, K. N., K. Pratis, M. E. Jones and E. R. Simpson (2004). "Estrogen replacement reverses the hepatic steatosis phenotype in the male aromatase knockout mouse." Endocrinology 145(4): 1842-1848.

Honda, S., N. Harada, S. Ito, Y. Takagi and S. Maeda (1998). "Disruption of sexual behavior in male aromatase-deficient mice lacking exons 1 and 2 of the cyp19 gene." Biochem Biophys Res Commun 252(2): 445-449.

Huang, S., X. Du, R. Wang, R. Li, H. Wang, L. Luo, S. O'Leary, J. Qiao and B. W. J. Mol (2018). "Ovulation induction and intrauterine insemination in infertile women with polycystic ovary syndrome: A comparison of drugs." Eur J Obstet Gynecol Reprod Biol 231: 117-121.

Hutchison, J. B. (1993). "Aromatase: neuromodulator in the control of behavior." J Steroid Biochem Mol Biol 44(4-6): 509-520.

Information-NCBI, N. C. f. B. from https://www.ncbi.nlm.nih.gov/.

Iorga, A., C. M. Cunningham, S. Moazeni, G. Ruffenach, S. Umar and M. Eghbali (2017). "The protective role of estrogen and estrogen receptors in cardiovascular disease and the controversial use of estrogen therapy." Biol Sex Differ 8(1): 33.

Ishunina, T. A., D. F. Fischer and D. F. Swaab (2007). "Estrogen receptor alpha and its splice variants in the hippocampus in aging and Alzheimer's disease." Neurobiol Aging 28(11): 1670-1681. Izumo, N., Y. Ishibashi, M. Ohba, T. Morikawa and T. Manabe (2012). "Decreased voluntary activity and amygdala levels of serotonin and dopamine in ovariectomized rats." Behav Brain Res 227(1): 1-6.

Jardi, F., M. R. Laurent, N. Kim, R. Khalil, D. De Bundel, A. Van Eeckhaut, L. Van Helleputte, L. Deboel, V. Dubois, D. Schollaert, B. Decallonne, G. Carmeliet, L. Van den Bosch, R. D'Hooge, F. Claessens and D. Vanderschueren (2018). "Testosterone boosts physical activity in male mice via dopaminergic pathways." Sci Rep 8(1): 957.

Jasarevic, E., P. T. Sieli, E. E. Twellman, T. H. Welsh, Jr., T. R. Schachtman, R. M. Roberts, D. C. Geary and C. S. Rosenfeld (2011). "Disruption of adult expression of sexually selected traits by developmental exposure to bisphenol A." Proc Natl Acad Sci U S A 108(28): 11715-11720.

Jenkins, V., V. Shilling, L. Fallowfield, A. Howell and S. Hutton (2004). "Does hormone therapy for the treatment of breast cancer have a detrimental effect on memory and cognition? A pilot study." Psychooncology 13(1): 61-66.

86 Jones, M. E., W. C. Boon, J. Proietto and E. R. Simpson (2006). "Of mice and men: the evolving phenotype of aromatase deficiency." Trends Endocrinol Metab 17(2): 55-64.

Jones, M. E., A. W. Thorburn, K. L. Britt, K. N. Hewitt, N. G. Wreford, J. Proietto, O. K. Oz, B. J. Leury, K. M. Robertson, S. Yao and E. R. Simpson (2000). "Aromatase-deficient (ArKO) mice have a phenotype of increased adiposity." Proc Natl Acad Sci U S A 97(23): 12735-12740.

Jurka, J., V. V. Kapitonov, A. Pavlicek, P. Klonowski, O. Kohany and J. Walichiewicz (2005). "Repbase Update, a database of eukaryotic repetitive elements." Cytogenet Genome Res 110(1-4): 462-467.

Kadioglu, P., G. Oral, M. Sayitoglu, N. Erensoy, B. Senel, N. Gazioglu, A. Sav, G. Cetin and U. Ozbek (2008). "Aromatase cytochrome P450 enzyme expression in human pituitary." Pituitary 11(1): 29-35.

Klinker, F., K. Kohnemann, W. Paulus and D. Liebetanz (2017). "Dopamine D3 receptor status modulates sexual dimorphism in voluntary wheel running behavior in mice." Behav Brain Res 333: 235-241.

Komiya, Y. and R. Habas (2008). "Wnt signal transduction pathways." Organogenesis 4(2): 68-75.

Konkle, A. T. and J. Balthazart (2011). "Sex differences in the rapid control of aromatase activity in the quail preoptic area." J Neuroendocrinol 23(5): 424-434.

Korner, G., T. Scherer, D. Adamsen, A. Rebuffat, M. Crabtree, A. Rassi, R. Scavelli, D. Homma, B. Ledermann, D. Konrad, H. Ichinose, C. Wolfrum, M. Horsch, B. Rathkolb, M. Klingenspor, J. Beckers, E. Wolf, V. Gailus-Durner, H. Fuchs, M. Hrabe de Angelis, N. Blau, J. Rozman and B. Thony (2016). "Mildly compromised tetrahydrobiopterin cofactor biosynthesis due to Pts variants leads to unusual body fat distribution and abdominal obesity in mice." J Inherit Metab Dis 39(2): 309-319.

Kramer, K. M., A. N. Perry, D. Golbin and B. S. Cushing (2009). "Sex steroids are necessary in the second postnatal week for the expression of male alloparental behavior in prairie voles (Microtus ochragaster)." Behav Neurosci 123(5): 958- 963.

Kravitz, H. M., P. M. Meyer, T. E. Seeman, G. A. Greendale and M. R. Sowers (2006). "Cognitive functioning and sex steroid hormone gene polymorphisms in women at midlife." Am J Med 119(9 Suppl 1): S94-S102.

87 Kurian, J. R., K. M. Olesen and A. P. Auger (2010). "Sex differences in epigenetic regulation of the estrogen receptor-alpha promoter within the developing preoptic area." Endocrinology 151(5): 2297-2305.

Langmead, B., C. Trapnell, M. Pop and S. L. Salzberg (2009). "Ultrafast and memory-efficient alignment of short DNA sequences to the human genome." Genome Biol 10(3): R25.

Lavaque, E., A. Mayen, I. Azcoitia, M. Tena-Sempere and L. M. Garcia-Segura (2006). "Sex differences, developmental changes, response to injury and cAMP regulation of the mRNA levels of steroidogenic acute regulatory protein, cytochrome p450scc, and aromatase in the olivocerebellar system." J Neurobiol 66(3): 308-318.

Lee, J. R., K. E. Parker, M. Tapia, H. W. Johns, T. G. Floros, M. D. Roberts, F. W. Booth and M. J. Will (2019). "Voluntary wheel running effects on intra- accumbens opioid high-fat feeding and locomotor behavior in Sprague-Dawley and Wistar rat strains." Physiol Behav 206: 67-75.

Lein, E. S., M. J. Hawrylycz, N. Ao, M. Ayres, A. Bensinger, A. Bernard, A. F. Boe, M. S. Boguski, K. S. Brockway, E. J. Byrnes, L. Chen, L. Chen, T. M. Chen, M. C. Chin, J. Chong, B. E. Crook, A. Czaplinska, C. N. Dang, S. Datta, N. R. Dee, A. L. Desaki, T. Desta, E. Diep, T. A. Dolbeare, M. J. Donelan, H. W. Dong, J. G. Dougherty, B. J. Duncan, A. J. Ebbert, G. Eichele, L. K. Estin, C. Faber, B. A. Facer, R. Fields, S. R. Fischer, T. P. Fliss, C. Frensley, S. N. Gates, K. J. Glattfelder, K. R. Halverson, M. R. Hart, J. G. Hohmann, M. P. Howell, D. P. Jeung, R. A. Johnson, P. T. Karr, R. Kawal, J. M. Kidney, R. H. Knapik, C. L. Kuan, J. H. Lake, A. R. Laramee, K. D. Larsen, C. Lau, T. A. Lemon, A. J. Liang, Y. Liu, L. T. Luong, J. Michaels, J. J. Morgan, R. J. Morgan, M. T. Mortrud, N. F. Mosqueda, L. L. Ng, R. Ng, G. J. Orta, C. C. Overly, T. H. Pak, S. E. Parry, S. D. Pathak, O. C. Pearson, R. B. Puchalski, Z. L. Riley, H. R. Rockett, S. A. Rowland, J. J. Royall, M. J. Ruiz, N. R. Sarno, K. Schaffnit, N. V. Shapovalova, T. Sivisay, C. R. Slaughterbeck, S. C. Smith, K. A. Smith, B. I. Smith, A. J. Sodt, N. N. Stewart, K. R. Stumpf, S. M. Sunkin, M. Sutram, A. Tam, C. D. Teemer, C. Thaller, C. L. Thompson, L. R. Varnam, A. Visel, R. M. Whitlock, P. E. Wohnoutka, C. K. Wolkey, V. Y. Wong, M. Wood, M. B. Yaylaoglu, R. C. Young, B. L. Youngstrom, X. F. Yuan, B. Zhang, T. A. Zwingman and A. R. Jones (2007). "Genome-wide atlas of gene expression in the adult mouse brain." Nature 445(7124): 168-176.

Ley, C. J., B. Lees and J. C. Stevenson (1992). "Sex- and menopause-associated changes in body-fat distribution." Am J Clin Nutr 55(5): 950-954. Li, C., C. Zhou and R. Li (2016). "Can Exercise Ameliorate Aromatase Inhibitor- Induced Cognitive Decline in Breast Cancer Patients?" Mol Neurobiol 53(6): 4238-4246.

88 Liew, S. H., A. E. Drummond, M. E. Jones and J. K. Findlay (2010). "The lack of estrogen and excess luteinizing hormone are responsible for the female ArKO mouse phenotype." Mol Cell Endocrinol 327(1-2): 56-64.

Lonstein, J. S. and G. J. De Vries (2000). "Influence of gonadal hormones on the development of parental behavior in adult virgin prairie voles (Microtus ochrogaster)." Behav Brain Res 114(1-2): 79-87.

Lonstein, J. S. and G. J. De Vries (2000). "Sex differences in the parental behavior of rodents." Neurosci Biobehav Rev 24(6): 669-686.

Love, M. I., W. Huber and S. Anders (2014). "Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2." Genome Biol 15(12): 550.

Lynch, J. F., 3rd, D. Dejanovic, P. Winiecki, J. Mulvany, S. Ortiz, D. C. Riccio and A. M. Jasnow (2014). "Activation of ERbeta modulates fear generalization through an effect on memory retrieval." Horm Behav 66(2): 421-429.

Lynch, J. F., 3rd, T. Vanderhoof, P. Winiecki, M. S. Latsko, D. C. Riccio and A. M. Jasnow (2016). "Aromatized testosterone attenuates contextual generalization of fear in male rats." Horm Behav 84: 127-135.

MacDonald, T. L., K. L. Ritchie, S. Davies, M. J. Hamilton, D. T. Cervone and D. J. Dyck (2015). "Exercise training is an effective alternative to estrogen supplementation for improving glucose homeostasis in ovariectomized rats." Physiol Rep 3(11).

Martin, M. (2011). "Cutadapt removes adaptor sequences from high-throughput sequencing reads." EMBnet.journal 17: 10.

Martin, S., M. Jones, E. Simpson and M. van den Buuse (2003). "Impaired spatial reference memory in aromatase-deficient (ArKO) mice." Neuroreport 14(15): 1979-1982.

Mathes, W. F., D. L. Nehrenberg, R. Gordon, K. Hua, T. Garland, Jr. and D. Pomp (2010). "Dopaminergic dysregulation in mice selectively bred for excessive exercise or obesity." Behav Brain Res 210(2): 155-163.

Matsuda, K. I., H. Mori, B. M. Nugent, D. W. Pfaff, M. M. McCarthy and M. Kawata (2011). "Histone deacetylation during brain development is essential for permanent masculinization of sexual behavior." Endocrinology 152(7): 2760- 2767.

McAllister, C., J. Long, A. Bowers, A. Walker, P. Cao, S. Honda, N. Harada, M. Staufenbiel, Y. Shen and R. Li (2010). "Genetic targeting aromatase in male

89 amyloid precursor protein transgenic mice down-regulates beta-secretase (BACE1) and prevents Alzheimer-like pathology and cognitive impairment." J Neurosci 30(21): 7326-7334.

McCarthy, M. M., B. M. Nugent and K. M. Lenz (2017). "Neuroimmunology and neuroepigenetics in the establishment of sex differences in the brain." Nat Rev Neurosci 18(8): 471-484.

Medway, C., O. Combarros, M. Cortina-Borja, H. T. Butler, C. A. Ibrahim- Verbaas, R. F. de Bruijn, P. J. Koudstaal, C. M. van Duijn, M. A. Ikram, I. Mateo, P. Sanchez-Juan, M. G. Lehmann, R. Heun, H. Kolsch, P. Deloukas, N. Hammond, E. Coto, V. Alvarez, P. G. Kehoe, R. Barber, G. K. Wilcock, K. Brown, O. Belbin, D. R. Warden, A. D. Smith, K. Morgan and D. J. Lehmann (2014). "The sex-specific associations of the aromatase gene with Alzheimer's disease and its interaction with IL10 in the Epistasis Project." Eur J Hum Genet 22(2): 216-220.

Melcangi, R. C., S. Giatti and L. M. Garcia-Segura (2016). "Levels and actions of neuroactive steroids in the nervous system under physiological and pathological conditions: Sex-specific features." Neurosci Biobehav Rev 67: 25-40. Meng, F. T., R. J. Ni, Z. Zhang, J. Zhao, Y. J. Liu and J. N. Zhou (2011). "Inhibition of oestrogen biosynthesis induces mild anxiety in C57BL/6J ovariectomized female mice." Neurosci Bull 27(4): 241-250.

Meredith, G. E., B. A. Baldo, M. E. Andrezjewski and A. E. Kelley (2008). "The structural basis for mapping behavior onto the ventral striatum and its subdivisions." Brain Struct Funct 213(1-2): 17-27.

Mouser, J. G., P. D. Loprinzi and J. P. Loenneke (2016). "The association between physiologic testosterone levels, lean mass, and fat mass in a nationally representative sample of men in the United States." Steroids 115: 62-66.

Mukaka, M. M. (2012). "Statistics corner: A guide to appropriate use of correlation coefficient in medical research." Malawi Med J 24(3): 69-71.

Muller, S. T., A. M. Keiler, K. Kraker, O. Zierau and R. Bernhardt (2018). "Influence of estrogen on individual exercise motivation and bone protection in ovariectomized rats." Lab Anim: 23677218756455.

Murray, E. K., A. Hien, G. J. de Vries and N. G. Forger (2009). "Epigenetic control of sexual differentiation of the bed nucleus of the stria terminalis." Endocrinology 150(9): 4241-4247.

Naftolin, F. (1994). "Brain aromatization of androgens." J Reprod Med 39(4): 257261.

90 Naftolin, F., K. J. Ryan, I. J. Davies, V. V. Reddy, F. Flores, Z. Petro, M. Kuhn, R. J. White, Y. Takaoka and L. Wolin (1975). "The formation of estrogens by central neuroendocrine tissues." Recent Prog Horm Res 31: 295-319.

Naftolin, F., K. J. Ryan and Z. Petro (1971). "Aromatization of androstenedione by limbic system tissue from human foetuses." J Endocrinol 51(4): 795-796.

Naftolin, F., K. J. Ryan and Z. Petro (1972). "Aromatization of androstenedione by the anterior hypothalamus of adult male and female rats." Endocrinology 90(1): 295-298.

Nelson, L. R. and S. E. Bulun (2001). "Estrogen production and action." J Am Acad Dermatol 45(3 Suppl): S116-124.

Nemoto, Y., K. Toda, M. Ono, K. Fujikawa-Adachi, T. Saibara, S. Onishi, H. Enzan, T. Okada and Y. Shizuta (2000). "Altered expression of fatty acid- metabolizing enzymes in aromatase-deficient mice." J Clin Invest 105(12): 1819- 1825. Nugent, B. M., C. L. Wright, A. C. Shetty, G. E. Hodes, K. M. Lenz, A. Mahurkar, S. J. Russo, S. E. Devine and M. M. McCarthy (2015). "Brain feminization requires active repression of masculinization via DNA methylation." Nat Neurosci 18(5): 690-697.

Ohlsson, C., A. Hammarstedt, L. Vandenput, N. Saarinen, H. Ryberg, S. H. Windahl, H. H. Farman, J. O. Jansson, S. Moverare-Skrtic, U. Smith, F. P. Zhang, M. Poutanen, S. Hedjazifar and K. Sjogren (2017). "Increased adipose tissue aromatase activity improves insulin sensitivity and reduces adipose tissue inflammation in male mice." Am J Physiol Endocrinol Metab 313(4): E450-E462.

Op de Macks, Z. A., S. A. Bunge, O. N. Bell, L. Wilbrecht, L. J. Kriegsfeld, A. S. Kayser and R. E. Dahl (2016). "Risky decision-making in adolescent girls: The role of pubertal hormones and reward circuitry." Psychoneuroendocrinology 74: 77-91.

Ortega, M. T., N. J. Bivens, T. Jogahara, A. Kuroiwa, S. A. Givan and C. S. Rosenfeld (2019). "Sexual dimorphism in brain transcriptomes of Amami spiny rats (Tokudaia osimensis): a rodent species where males lack the Y chromosome." BMC Genomics 20(1): 87.

Padilla, J., N. T. Jenkins, M. D. Roberts, A. A. Arce-Esquivel, J. S. Martin, M. H. Laughlin and F. W. Booth (2013). "Differential changes in vascular mRNA levels between rat iliac and renal arteries produced by cessation of voluntary running." Exp Physiol 98(1): 337-347.

91 Park, S. H., M. Tish Knobf and S. Jeon (2019). "Endocrine Therapy-Related Symptoms and Quality of Life in Female Cancer Survivors in the Yale Fitness Intervention Trial." J Nurs Scholarsh 51(3): 317-325.

Park, Y. M., J. A. Kanaley, J. Padilla, T. Zidon, R. J. Welly, M. J. Will, S. L. Britton, L. G. Koch, G. N. Ruegsegger, F. W. Booth, J. P. Thyfault and V. J. Vieira-Potter (2016). "Effects of intrinsic aerobic capacity and ovariectomy on voluntary wheel running and nucleus accumbens dopamine receptor gene expression." Physiol Behav 164(Pt A): 383-389.

Patel, S. (2017). "Disruption of aromatase homeostasis as the cause of a multiplicity of ailments: A comprehensive review." J Steroid Biochem Mol Biol 168: 19-25.

Perez-Leighton, C., M. R. Little, M. Grace, C. Billington and C. M. Kotz (2017). "Orexin signaling in rostral lateral hypothalamus and nucleus accumbens shell in the control of spontaneous physical activity in high- and low-activity rats." Am J Physiol Regul Integr Comp Physiol 312(3): R338-R346. Peterson, B. M., P. G. Mermelstein and R. L. Meisel (2015). "Estradiol mediates dendritic spine plasticity in the nucleus accumbens core through activation of mGluR5." Brain Struct Funct 220(4): 2415-2422.

Pietranera, L., M. E. Brocca, P. Roig, A. Lima, L. M. Garcia-Segura and A. F. De Nicola (2015). "Estrogens are neuroprotective factors for hypertensive encephalopathy." J Steroid Biochem Mol Biol 146: 15-25.

Raudvere, U., L. Kolberg, I. Kuzmin, T. Arak, P. Adler, H. Peterson and J. Vilo (2019). "g:Profiler: a web server for functional enrichment analysis and conversions of gene lists (2019 update)." Nucleic Acids Res.

Reddy, R. C., C. T. Estill, M. Meaker, F. Stormshak and C. E. Roselli (2014). "Sex differences in expression of oestrogen receptor alpha but not androgen receptor mRNAs in the foetal lamb brain." J Neuroendocrinol 26(5): 321-328.

Roberts, M. D., L. Gilpin, K. E. Parker, T. E. Childs, M. J. Will and F. W. Booth (2012). "Dopamine D1 receptor modulation in nucleus accumbens lowers voluntary wheel running in rats bred to run high distances." Physiol Behav 105(3): 661-668.

Roberts, M. D., G. N. Ruegsegger, J. D. Brown and F. W. Booth (2017). "Mechanisms Associated With Physical Activity Behavior: Insights From Rodent Experiments." Exerc Sport Sci Rev 45(4): 217-222.

Roseguini, B. T., S. Mehmet Soylu, J. J. Whyte, H. T. Yang, S. Newcomer and M. H. Laughlin (2010). "Intermittent pneumatic leg compressions acutely

92 upregulate VEGF and MCP-1 expression in skeletal muscle." Am J Physiol Heart Circ Physiol 298(6): H1991-2000.

Roselli, C. E., F. Stormshak and J. A. Resko (1998). "Distribution and regulation of aromatase activity in the ram hypothalamus and amygdala." Brain Res 811(1- 2): 105-110.

Rosenfeld, C. S. (2017). "Sex-dependent differences in voluntary physical activity." J Neurosci Res 95(1-2): 279-290.

Rosenfeld, C. S. and S. A. Ferguson (2014). "Barnes maze testing strategies with small and large rodent models." J Vis Exp(84): e51194.

Ruegsegger, G. N., J. D. Brown, M. C. Kovarik, D. K. Miller and F. W. Booth (2016). "Mu-opioid receptor inhibition decreases voluntary wheel running in a dopamine-dependent manner in rats bred for high voluntary running." Neuroscience 339: 525-537. Ruegsegger, G. N., R. G. Toedebusch, T. E. Childs, K. B. Grigsby and F. W. Booth (2017). "Loss of Cdk5 function in the nucleus accumbens decreases wheel running and may mediate age-related declines in voluntary physical activity." J Physiol 595(1): 363-384.

Ruegsegger, G. N., R. G. Toedebusch, M. J. Will and F. W. Booth (2015). "Mu opioid receptor modulation in the nucleus accumbens lowers voluntary wheel running in rats bred for high running motivation." Neuropharmacology 97: 171- 181.

Russell, N. and M. Grossmann (2019). "MECHANISMS IN ENDOCRINOLOGY: Estradiol as a male hormone." Eur J Endocrinol.

Sasano, H., K. Takashashi, F. Satoh, H. Nagura and N. Harada (1998). "Aromatase in the human central nervous system." Clin Endocrinol (Oxf) 48(3): 325-329.

Satta, R., B. Certa, D. He and A. W. Lasek (2018). "Estrogen Receptor beta in the Nucleus Accumbens Regulates the Rewarding Properties of Cocaine in Female Mice." Int J Neuropsychopharmacol 21(4): 382-392.

Schwarz, J. M., B. M. Nugent and M. M. McCarthy (2010). "Developmental and hormone-induced epigenetic changes to estrogen and progesterone receptor genes in brain are dynamic across the life span." Endocrinology 151(10): 4871-4881.

Shuling, L., M. L. Sie Kuei, S. E. Saffari, Z. Jiayun, T. T. Yeun, J. P. W. Leng, V. Viardot-Foucault, S. Nadarajah, J. K. Y. Chan and T. H. Hao (2019). "Do men with normal testosterone-oestradiol ratios benefit from letrozole for the treatment of male infertility?" Reprod Biomed Online 38(1): 39-45.

93

Simpson, E. R. (2000). "Role of aromatase in sex steroid action." J Mol Endocrinol 25(2): 149-156.

Simpson, E. R., C. Clyne, G. Rubin, W. C. Boon, K. Robertson, K. Britt, C. Speed and M. Jones (2002). "Aromatase--a brief overview." Annu Rev Physiol 64: 93-127.

Simpson, E. R., Y. Zhao, V. R. Agarwal, M. D. Michael, S. E. Bulun, M. M. Hinshelwood, S. Graham-Lorence, T. Sun, C. R. Fisher, K. Qin and C. R. Mendelson (1997). "Aromatase expression in health and disease." Recent Prog Horm Res 52: 185-213; discussion 213-184.

Staffend, N. A., C. M. Loftus and R. L. Meisel (2011). "Estradiol reduces dendritic spine density in the ventral striatum of female Syrian hamsters." Brain Struct Funct 215(3-4): 187-194.

Starkey, N. J. E., Y. Li, S. K. Drenkhahn-Weinaug, J. Liu and D. B. Lubahn (2018). "27-Hydroxycholesterol Is an Estrogen Receptor beta-Selective Negative Allosteric Modifier of 17beta-Estradiol Binding." Endocrinology 159(5): 1972- 1981.

Stefanska, A., K. Bergmann and G. Sypniewska (2015). "Metabolic Syndrome and Menopause: Pathophysiology, Clinical and Diagnostic Significance." Adv Clin Chem 72: 1-75.

Stelzer, G., N. Rosen, I. Plaschkes, S. Zimmerman, M. Twik, S. Fishilevich, T. I. Stein, R. Nudel, I. Lieder, Y. Mazor, S. Kaplan, D. Dahary, D. Warshawsky, Y. Guan-Golan, A. Kohn, N. Rappaport, M. Safran and D. Lancet (2016). "The GeneCards Suite: From Gene Data Mining to Disease Genome Sequence Analyses." Curr Protoc Bioinformatics 54: 1 30 31-31 30 33.

Stoffel-Wagner, B., M. Watzka, J. Schramm, F. Bidlingmaier and D. Klingmuller (1999). "Expression of CYP19 (aromatase) mRNA in different areas of the human brain." J Steroid Biochem Mol Biol 70(4-6): 237-241.

Svendsen, O. L., C. Hassager and C. Christiansen (1995). "Age- and menopause- associated variations in body composition and fat distribution in healthy women as measured by dual-energy X-ray absorptiometry." Metabolism 44(3): 369-373.

System, G. O. (2019). "GNU Bash." Retrieved 22nd April 2016, from https://www.gnu.org/software/bash/.

Szklarczyk, D., A. L. Gable, D. Lyon, A. Junge, S. Wyder, J. Huerta-Cepas, M. Simonovic, N. T. Doncheva, J. H. Morris, P. Bork, L. J. Jensen and C. V. Mering (2019). "STRING v11: protein-protein association networks with increased

94 coverage, supporting functional discovery in genome-wide experimental datasets." Nucleic Acids Res 47(D1): D607-D613.

Takahashi, K., T. Hosoya, K. Onoe, H. Doi, H. Nagata, T. Hiramatsu, X. L. Li, Y. Watanabe, Y. Wada, T. Takashima, M. Suzuki, H. Onoe and Y. Watanabe (2014). "11C-cetrozole: an improved C-11C-methylated PET probe for aromatase imaging in the brain." J Nucl Med 55(5): 852-857.

Takeda, K., K. Toda, T. Saibara, M. Nakagawa, K. Saika, T. Onishi, T. Sugiura and Y. Shizuta (2003). "Progressive development of insulin resistance phenotype in male mice with complete aromatase (CYP19) deficiency." J Endocrinol 176(2): 237-246.

Thomas, G. A., B. Cartmel, M. Harrigan, M. Fiellin, S. Capozza, Y. Zhou, E. Ercolano, C. P. Gross, D. Hershman, J. Ligibel, K. Schmitz, F. Y. Li, T. Sanft and M. L. Irwin (2017). "The effect of exercise on body composition and bone mineral density in breast cancer survivors taking aromatase inhibitors." Obesity (Silver Spring) 25(2): 346-351.

Thornton, M. J., L. D. Nelson, A. H. Taylor, M. P. Birch, I. Laing and A. G. Messenger (2006). "The modulation of aromatase and estrogen receptor alpha in cultured human dermal papilla cells by dexamethasone: a novel mechanism for selective action of estrogen via estrogen receptor beta?" J Invest Dermatol 126(9): 2010-2018.

Thorpe, A. J. and C. M. Kotz (2005). "Orexin A in the nucleus accumbens stimulates feeding and locomotor activity." Brain Res 1050(1-2): 156-162.

Tobiansky, D. J., A. M. Korol, C. Ma, J. E. Hamden, C. Jalabert, R. J. Tomm and K. K. Soma (2018). "Testosterone and Corticosterone in the Mesocorticolimbic System of Male Rats: Effects of Gonadectomy and Caloric Restriction." Endocrinology 159(1): 450-464.

Toda, K., K. Takeda, T. Okada, S. Akira, T. Saibara, T. Kaname, K. Yamamura, S. Onishi and Y. Shizuta (2001). "Targeted disruption of the aromatase P450 gene (Cyp19) in mice and their ovarian and uterine responses to 17beta-oestradiol." J Endocrinol 170(1): 99-111.

Tonn Eisinger, K. R., K. S. Gross, B. P. Head and P. G. Mermelstein (2018). "Interactions between estrogen receptors and metabotropic glutamate receptors and their impact on drug addiction in females." Horm Behav. Tonn Eisinger, K. R., E. B. Larson, M. I. Boulware, M. J. Thomas and P. G. Mermelstein (2018). "Membrane estrogen receptor signaling impacts the reward circuitry of the female brain to influence motivated behaviors." Steroids 133: 53- 59.

95 UniProt, C. (2019). "UniProt: a worldwide hub of protein knowledge." Nucleic Acids Res 47(D1): D506-D515.

Vieira-Potter, V. J., T. L. Cross, K. S. Swanson, S. J. Sarma, Z. Lei, L. W. Sumner and C. S. Rosenfeld (2018). "Soy-Induced Fecal Metabolome Changes in Ovariectomized and Intact Female Rats: Relationship with Cardiometabolic Health." Sci Rep 8(1): 16896. Vieira-Potter, V. J., J. Padilla, Y. M. Park, R. J. Welly, R. J. Scroggins, S. L. Britton, L. G. Koch, N. T. Jenkins, J. M. Crissey, T. Zidon, E. M. Morris, G. M. Meers and J. P. Thyfault (2015). "Female rats selectively bred for high intrinsic aerobic fitness are protected from ovariectomy-associated metabolic dysfunction." Am J Physiol Regul Integr Comp Physiol 308(6): R530-542.

Vierk, R., J. Bayer, S. Freitag, M. Muhia, K. Kutsche, T. Wolbers, M. Kneussel, T. Sommer and G. M. Rune (2015). "Structure-function-behavior relationship in estrogen-induced synaptic plasticity." Horm Behav 74: 139-148.

Wade, J. (2001). "Zebra finch sexual differentiation: the aromatization hypothesis revisited." Microsc Res Tech 54(6): 354-363.

West, E. A. and R. M. Carelli (2016). "Nucleus Accumbens Core and Shell Differentially Encode Reward-Associated Cues after Reinforcer Devaluation." J Neurosci 36(4): 1128-1139.

Wissman, A. M., A. F. McCollum, G. Z. Huang, A. A. Nikrodhanond and C. S. Woolley (2011). "Sex differences and effects of cocaine on excitatory synapses in the nucleus accumbens." Neuropharmacology 61(1-2): 217-227.

Wright, C. L., J. S. Schwarz, S. L. Dean and M. M. McCarthy (2010). "Cellular mechanisms of estradiol-mediated sexual differentiation of the brain." Trends Endocrinol Metab 21(9): 553-561.

Yague, J. G., A. Munoz, P. de Monasterio-Schrader, J. Defelipe, L. M. Garcia- Segura and I. Azcoitia (2006). "Aromatase expression in the human temporal cortex." Neuroscience 138(2): 389-401.

Zhan, T., N. Rindtorff and M. Boutros (2017). "Wnt signaling in cancer." Oncogene 36(11): 1461-1473.

Zhang, J., Z. X. He, L. M. Wang, W. Yuan, L. F. Li, W. J. Hou, Y. Yang, Q. Q. Guo, X. N. Zhang, W. Q. Cai, S. C. An and F. D. Tai (2019). "Voluntary Wheel Running Reverses Deficits in Social Behavior Induced by Chronic Social Defeat Stress in Mice: Involvement of the Dopamine System." Front Neurosci 13: 256.

Zhang, Q. G., R. Wang, H. Tang, Y. Dong, A. Chan, G. R. Sareddy, R. K. Vadlamudi and D. W. Brann (2014). "Brain-derived estrogen exerts anti-

96 inflammatory and neuroprotective actions in the rat hippocampus." Mol Cell Endocrinol 389(1-2): 84-91.

Zhu, X., D. Ottenheimer and R. J. DiLeone (2016). "Activity of D1/2 Receptor Expressing Neurons in the Nucleus Accumbens Regulates Running, Locomotion, and Food Intake." Front Behav Neurosci 10: 66.

Zhuang, P. C., Z. N. Tan, Z. Y. Jia, B. Wang, J. J. Grady and X. M. Ma (2019). "Treadmill Exercise Reverses Depression Model-Induced Alteration of Dendritic Spines in the Brain Areas of Mood Circuit." Front Behav Neurosci 13: 93.

97