A Dissertation

entitled

Hypothalamic Melanocortin 4 Receptors Regulate Sexual Behavior in Mice

by

Erin Semple

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the

Doctor of Philosophy Degree in

Biomedical Sciences

______Jennifer Hill, PhD, Committee Chair

______David Giovannucci, PhD, Committee Member

______Joshua Park, PhD, Committee Member

______Edwin Sanchez, PhD, Committee Member

______Ruili Xie, PhD, Committee Member

______Amanda Bryant-Friedrich, Dr. rer Nat., Dean College of Graduate Studies

The University of Toledo

August 2017

Copyright 2017, Erin A. Semple

This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of

Hypothalamic Melanocortin 4 Receptors Regulate Sexual Behavior in Mice

by

Erin Semple

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biomedical Sciences

The University of Toledo

August 2017

Sexual dysfunction affects approximately one third of men and 40% of women in the United States and world-wide. Unfortunately, existing treatments that target the periphery are not effective for all types of sexual dysfunction, and the involvement of the central nervous system in sexual behavior is not well-understood. Preclinical data has shown that central melanocortins and their receptors, particularly the melanocortin 4 receptor (MC4R), may play a role in sexual behavior in both male and female rodents.

We found that six-month-old male MC4R null mice showed evidence of erectile dysfunction and an inability to ejaculate. Due to the high expression of MC4R in the paraventricular nucleus of the hypothalamus (PVN), we hypothesized that the PVN is a key site of melanocortin-mediated sexual behavior. Using Sim1 as a target for the PVN, we tested the sexual behavior of a tbMC4Rsim1 transgenic mouse model in which MC4R was only expressed on Sim1 neurons. These mice did not have the sexual impairments seen in MC4RKO mice, implying that MC4R on Sim1 neurons is sufficient for erectile function and ejaculation. To reduce the confound of age-related obesity in MC4RKO mice, we also tested these mice at two-months of age. The younger MC4RKO mice had a different phenotype, with the only sexual deficit being delayed ejaculation. This was also iii recovered in tbMC4Rsim1 mice. Furthermore, expressing MC4R only on oxytocin neurons similarly recovered the sexual impairment in MC4R null males. Finally, a metabolic profile indicated that the delayed ejaculation seen in MC4RKO mice at two months of age was independent of MC4R-mediated weight gain. Female MC4RKO mice had a decreased lordosis quotient at two months of age. Expression of MC4R only on Sim1 neurons or oxytocin neurons resulted in a lordosis quotient comparable to controls, despite a similar metabolic profile to MC4R null mice. This study implicates MC4R on

Sim1 neurons, and more specifically oxytocin neurons, in the central neurocircuitry underlying sexual behavior in both male and female mice.

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Acknowledgements

I would like to acknowledge my advisor, Dr. Jennifer Hill, for providing me with the opportunity to work in her lab. Without her guidance and support, none of this research would have been possible. I am also grateful for the constant advice, support, and companionship of the members of the Hill lab who made my graduate studies enjoyable.

I would also like to thank my committee members, the faculty, staff, and students of the Department of Neurosciences as well as the Department of Physiology and

Pharmacology, and my fellow MD/PhD students who have provided me with a nurturing environment throughout my time in graduate school.

Finally, I would like to acknowledge my family and friends whose constant support and encouragement was invaluable. I owe a special shout-out to my significant other, Bradley, for having my back through all of the highs and lows of both medical school and graduate school.

Table of Contents

Abstract ...... iii

Acknowledgements ...... v

Table of Contents ...... vi

List of Figures ...... viii

List of Abbreviations ...... x

1 Challenges in Understanding Sexual Dysfunction ...... 1

1.1 Prevalence of Sexual Dysfunction ...... 1

1.2 Challenges in Definition ...... 3

1.2.1 Definitions for Men...... 3

1.2.2 Definitions for Women ...... 5

1.3 Comorbidities ...... 6

1.4 Treatment Options ...... 8

1.3.1 Treatments for Men...... 8

1.3.2 Treatments for Women ...... 9

1.5 Animal Models of Sexual Dysfunction...... 10

1.6 Central Control of Sexual Function ...... 12

1.7 The Melanocortin System and Sexual Dysfunction...... 13

1.8 Study Objectives ...... 15

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2 Sim1 Neurons are Sufficient for MC4R-Mediated Sexual Function in Six-Month-

Old Male Mice ...... 18

2.1 Introduction ...... 18

2.2 Methods…...... 20

2.3 Results…...... 23

2.4 Discussion ...... 27

2.5 Figures…...... 33

3 Hypothalamic Melanocortin 4 Receptors Influence Male Sexual

Behavior in Young Mice ...... 38

3.1 Introduction ...... 38

3.2 Methods…...... 40

3.3 Results…...... 44

3.4 Discussion ...... 51

3.5 Figures…...... 56

4 Hypothalamic Melanocortin 4 Receptors Affect Lordosis in Female Mice

Independent of Metabolic Changes ...... 70

4.1 Introduction ...... 70

4.2 Methods…...... 72

4.3 Results…...... 76

4.4 Discussion ...... 79

4.5 Figures…...... 84

5 Summary of Findings and Future Directions ...... 94

References ...... 99

vi

Chapter 1 ...... 99

Chapter 2 ...... 109

Chapter 3 ...... 113

Chapter 4 ...... 118

Chapter 5 ...... 124

vii

List of Figures

2-1 Sexual behavior in MC4RKO and tbMC4Rsim1 mice ...... 33

2-2 Sexual motivation was partially impaired by knocking out MC4R...... 34

2-3 Grooming induced by ICV αMSH requires MC4Rs on Sim1 neurons...... 35

2-4 ICV aMSH had an effect on tbMC4Rsim1 behavior ...... 36

2-5 Schematic of interpretation of results...... 37

3-1 Sexual behavior in MC4RKO mice...... 57

3-2 Sexual motivation in MC4RKO mice is unimpaired ...... 58

3-3 Colocalization of Sim1-cre with MC4R in Sim1-cre mice ...... 59

3-4 Colocalization of Sim1-cre with MC4R in tbMC4Rsim1 mice ...... 60

3-5 tbMC4Rsim1 mice had normal sexual behavior compared to controls...... 61

3-6 Colocalization of oxytocin neurons with MC4R...... 62

3-7 Sexual behavior of tbMC4Roxt was comparable to WT mice...... 63

3-8 MC4R has an effect on weight gain ...... 64

3-9 Metabolic cages normalized to lean body mass ...... 66

3-10 Activity, food intake, and water intake of male mice ...... 67

3-11 Sexual behavior of WT mice on high fat diet ...... 68

3-12 Serum hormones ...... 69

4-1 Lordosis Quotient of MC4RKO and tbMC4Rsim1 mice ...... 84

viii

4-2 Colocalization of Sim1-cre with MC4R...... 85

4-3 Oxytocin and MC4R are co-localized...... 86

4-4 Lordosis Quotient in tbMC4Roxt mice ...... 87

4-5 NMR in female mice ...... 88

4-6 GTT in female mice ...... 89

4-7 Metabolic cages in female mice ...... 90

4-8 Activity, food intake, and water intake of female mice ...... 91

4-9 Lordosis Quotient in high fat diet females ...... 92

4-10 Serum LH/FSH in females ...... 93

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List of Abbreviations

ARC ...... Arcuate Nucleus of the Hypothalamus ACTH ...... Adrenocorticotropic Hormone

BMI ...... Body Mass Index

DSM ...... Diagnostic Statistical Manual of Mental Disorders

ER ...... Estrogen Receptor

FSAD ...... Female Sexual Arousal Disorder FSIAD ...... Female Sexual Interest and Arousal Disorder FSH ...... Follicle-Stimulating Hormone

GTT ...... Glucose Tolerance Testing

HFD...... High Fat Diet HSDD ...... Hypoactive Sexual Desire Disorder

LH ...... Luteinizing Hormone

MC4R ...... Melanocortin 4 Receptor MC4RKO ...... Melanocortin 4 Receptor Knock-Out MPOA ...... Medial Preoptic Area MSH ...... Melanocyte Stimulating Hormone

NLOT ...... Nucleus of the Lateral Olfactory Tract NMR ...... Nuclear Magnetic Resonance NO ...... Nitric Oxide NTS ...... Nucleus Tractus Solitarius

PAG...... Periaqueductal Gray PDE-5 ...... Phosphodiesterase-5 PET ...... Positron Emission Tomography POMC ...... Proopiomelanocortin PVN...... Paraventricular Nucleus of the Hypothalamus

Sim1 ...... Single-minded Homolog 1 SON...... Supraoptic Nucleus

x

SSRI ...... Selective Serotonin Reuptake Inhibitor tbMC4Roxt ...... Re-expression of MC4R on oxytocin neurons tbMC4Rsim1 ...... Re-expression of MC4R on Sim1 neurons

VMH ...... Ventromedial Hypothalamus

WT ...... Wild-type

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Chapter 1

Challenges in Understanding Sexual Dysfunction

1.1 Prevalence of Sexual Dysfunction

Sexual Dysfunction is highly diagnosed in both men and women throughout the world. In the United States, about 30% of men and 40% of women experience some form of sexual dysfunction (73). These statistics have been relatively consistent around the world (33, 58, 75, 86, 95, 116) despite various cultural influences (66). Men and women of all ages report experiencing all forms of sexual dysfunction (73), although sexual function has been found to decline with age (81, 85), which may be partially due to increased life stressors (74) as well as biological influences (80). Survey results do not always reflect the decline in function with age, because older individuals may be less distressed by sexual dysfunction compared with their younger counterparts (41, 85).

Until recently, men have been the focus of sexual dysfunction studies. Statistics regarding specific types of sexual function have very high variability. Erectile dysfunction is the best studied form of sexual dysfunction, even though this is not usually reported as the most prevalent type of male sexual dysfunction. Studies report anywhere from 8% (116) to 22% (104) of men report being unable to maintain an erection, although rates increase dramatically when stratified by age (103). Issues with sexual interest have been reported at rates of approximately 5-18% of men (5, 116). With a prevalence of 30- 1

40%, ejaculation disorders are thought to be the most common form of sexual dysfunction, despite the lack of attention it receives compared to erectile dysfunction (2,

48, 103). Depending on the parameters used to obtain data, statistics on prevalence of premature ejaculation range from 13-30% of men (111, 116). Delayed or deficient ejaculation is not very well-understood, but only 1-4% of men are believed to have this disorder (1, 61). Similarly, delayed orgasm or anorgasmia causes great distress but is not very well-studied (62). Other forms of sexual dysfunction experienced by men that are less common include the inability to reach orgasm, delayed orgasm, not finding sex pleasurable, anxiety related to performing sexual activities, and pain during intercourse

(116).

Women in the United States have been found to have a prevalence of any sexual dysfunction to be approximately 40% (73). Other countries find similar rates of sexual dysfunction and sexual difficulties (20). One study of women in Australia found that 66% of over 1000 women reported experiencing at least one difficulty with sexual performance at baseline, with 36% of women experiencing at least one additional difficulty when asked 12 months later (115). Lack of interest or desire in sex has been found to reach a prevalence between 7 and 60% (20, 54, 114, 115). Difficulty with reaching orgasm is reported as the second most common with rates between 4 and 46%

(54, 115). Lack of interest in sex, as well as vaginal dryness, increased with age (115).

Sexual difficulties in women also increase in prevalence with age (20). Other sexual difficulties that women experience include reaching orgasm too quickly, not finding sex enjoyable, anxiety to engage in sex, and pain during intercourse (115).

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One of the challenges in interpreting the epidemiological data on female sexual dysfunction, is that there have been vast differences in data collection methodologies.

Some studies use interviews while others had participants fill out surveys. Studies vary widely in age, side effects, and even definitions of sexual phases and dysfunction (13, 14,

46, 55, 85).

1.2 Challenges in Definition

In order to obtain accurate epidemiology data, it is necessary to have adequate definitions for both normal and abnormal sexual function. Diagnoses depend heavily on both the definition of the disorder as well as the methods of obtaining statistics. The sharing of data is limited to the degree of which the definitions of the parameters are agreed upon. The sexual response cycle, first described by Masters and Johnson in 1966

(82), has undergone scrutiny over time, especially with regard to the cycle in women

(29). Definitions of the disorders in the Diagnostic Statistical Manual of Mental

Disorders (DSM) have evolved over time to try to reach more of a consensus (109).

Additionally, this lack in adequate definitions makes it hard to investigate treatment options, because these definitions are necessary for the development of appropriate outcome measures for the various aspects of sexual function.

1.2.1 Definitions for Men

Possibly due to the nature of the visibility of the male sexual anatomy, definitions for male sexual function have been more consistent across time. The sexual response cycle for both men and women begins with desire, or the expectation of engaging in sexual behavior. This progresses to arousal, which manifests as erection in men. The erection is maintained during the consummatory phase of the sexual pleasure cycle,

3

culminating in ejaculation and/or orgasm, which is followed be a refractory period (43).

Ejaculation occurs in two phases: emission and expulsion (113). The emission phase involves secretion of fluids and movement of these fluids to the proximal urethra, while expulsion is the muscle contraction that results in a release of these fluids (26). Sexual dysfunction in men is the inability of a man to successfully experience any of the phases of this sexual response cycle in the presence of distress over this inability (3). The

Diagnostic Statistical Manual (DSM) V defines the dysfunction of sexual desire as Male

Hypoactive Sexual Desire Disorder, and is defined as the persistent lack of sexual fantasies and desire for sexual activity (3, 84). The DSM V defines erectile disorder as the difficulty of obtaining an erection, maintaining an erection, or a general decrease in rigidity for almost all attempts at sexual intercourse (84). Premature ejaculation is defined as ejaculation about one minute after penetration contrary to the desires of the individual.

This must occur during most or all sexual occasions (3, 84). Notably, the one minute rule only applies to vaginal penetration and criteria have not been developed for other cases.

Delayed ejaculation is defined as an unwanted delay in ejaculation or absence of ejaculation in most or all occasions of sexual activity (84). It is important to highlight that these dysfunctions are only classified as a disorder if the individual experiences distress because of their inability to achieve any of these stages. This inability often causes distress in men who believe that successful intercourse is a representation of his masculinity (21, 92). The development of more specific definitions may help reduce stress in men who are not sure if their sexual function is considered normal.

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1.2.2 Definitions for Women

Definitions for normal sexual function have gone through many changes throughout history (4). Currently, the DSM V describes sexual dysfunction as a disturbance in any aspect of the sexual response cycle (3). The four stages of the sexual response cycle for women are often described in the same way as in men, using the stages of arousal, plateau, orgasm, and resolution, although other models have been proposed

(29, 44, 121). The DSM V combined the previously labeled Female Sexual Arousal

Disorder (FSAD) and Hypoactive Sexual Desire Disorder (HSDD) into one diagnosis called female sexual interest and arousal disorder (FSIAD) (121). Critics of this change in the DSM V suggest that while arousal and desire can both be affected in the same patient, but some patients are affected by only one or the other (31). Subsequently, combining the disorders into one may make it more difficult to accurately diagnose and treat a patient with only one of these disturbances. Other disorders recognized by the DSM V for women include orgasm disorders and pain disorders (3). Stigma also plays a role in sexual dysfunction in women. Women with more conservative beliefs regarding the

“sinful” nature of engaging in intercourse were more likely to experience Hyposexual

Desire Disorder while fear of sexual intercourse was a strong predictor of vaginismus

(91). More recently proposed models of the female sexual response cycle include aspects such as satisfaction with one’s own self-image, satisfaction with the relationship, and the influence of previous sexual experiences (29). The emphasis of these new models is that the goal of sex for females is personal satisfaction, rather than orgasm (12, 15, 29).

Placing emphasis on the psychological components of the sexual experience in both women and men have heavy implications for the treatment of sexual disorders (45).

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1.3 Co-morbidities

Sexual dysfunction is often found to be comorbid with other diagnoses. Sexual dysfunction in men and women have been found to be closely associated with cardiovascular disease (50, 90, 120). There is a particularly clear correlation between cardiovascular disease and erectile dysfunction, supporting the notion that this form of dysfunction is heavily influenced by peripheral disease. Similarly, men and women who have type 1 or type 2 diabetes are more likely to experience sexual dysfunction (34, 36,

77, 105, 131). Erectile dysfunction is up to four times more prevalent in men who have diabetes (9, 50). Up to 50% of men with type 2 diabetes reported dissatisfaction with their sexual lives (122).

The correlation between obesity and sexual dysfunction has been less clear.

Recent studies have found a link between erectile dysfunction and obesity (9, 50, 52), however, these studies were primarily done in men older than 40 years of age, who may have cardiovascular side-effects to obesity that may be more directly connected to sexual dysfunction. Importantly, men who altered their lifestyles to include exercise were able to improve erectile function (35). Obesity has not been shown to significantly affect sexual function in females (64, 71), although there is a negative correlation between BMI and orgasm as well as satisfaction (134).

There is an established association between erectile dysfunction and depression, but the nature of causality is unknown (50). Studies report that depression in the general population is more prevalent in men with erectile dysfunction compared to men without

(104), but it is unknown if men are more likely to be depressed because of their sexual dysfunction, or if depression actually leads to erectile dysfunction. Other studies show

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that both men and women who have depression have a higher prevalence of all types of sexual dysfunction compared to controls (10, 63). Use of certain anti-depressants, especially selective serotonin reuptake inhibitors (SSRIs), significantly increases the risk for erectile dysfunction in men and affects desire and orgasm in both men and women (9,

68).

Alcohol and nicotine use has had conflicting effects on sexual dysfunction. One study in men found no relationship between sexual dysfunction and alcohol or tobacco use (116), while another found that moderate drinking was negatively correlated with sexual dysfunction but smoking increased the risk for erectile dysfunction (9). Women who smoke have decreased interest in engaging in sexual activities (115). Alternatively, women who drink only once a week had less interest than women who drank more regularly (115).

Certain physical conditions have also been found to lead to sexual dysfunction, including lower urinary tract symptoms, chronic obstructive pulmonary disease, endocrine diseases, and benign prostatic hyperplasia (16, 50, 97, 125). Understanding how these comorbidities are associated with sexual function is important when considering treatment options for patients. In many of these cases, the comorbidity may be the underlying cause of the sexual dysfunction and focusing treatment on such a disease may return sexual function to normal. For example, anti-hypertensive medications used to treat hypertension have a positive effect on erectile dysfunction (32).

In the case of anti-depressants, however, sexual function can be negatively affected (68).

Understanding the pathways underlying both sexual dysfunction and related diseases will

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assist in the development and prescription of appropriate medications to meet the patient’s individual needs.

1.4 Treatment Options

1.4.1 Treatment for Men

The best known available treatments for sexual dysfunction are oral phosphodiesterase 5 (PDE-5) inhibitors for erectile dysfunction such as sildenafil, vardenafil, tadalafil, and avanafil (19). PDE-5 cleaves cGMP, which is stimulated by

Nitric Oxide (NO) to cause downstream smooth muscle cell relaxation in the corpora cavernosa of the penis. This process ends once cGMP is cleaved, but inhibitors that prevent PDE-5 from acting result in prolonged activity of cGMP on smooth muscle. This allows for blood to accumulate in the penis for appropriate rigidity to be achieved in erection (53). Because PDE-5 is found elsewhere in the periphery, common side effects include nasal congestion, dizziness, headaches, and flushing (53). In the case that PDE-5 inhibitors are ineffective, there are second-line treatments available for erectile dysfunction. Alprostadil, an intracavernosal injection that inhibits prostaglandin E1, also causes smooth muscle relaxation in the penis (53). These injections were found to be less effective in men with diabetes and metabolic syndrome (100). Papaverine works similarly alprostadil, and in combination with α-blockers such as phentolamine, there can be a substantial improvement in erectile function (53, 128). The primary issue with intracavernosal injection therapy is that the method of administration is unappealing to many patients and in those with prolonged use may have fibrosis of the corpus cavernosum or pain (56). Due to these issues, intraurethral and topical alprostadil have been developed as alternatives (53). All drugs that are currently approved for erectile

8

dysfunction target peripheral mechanisms, although drugs that target the central nervous system have been investigated.

Treatments for sexual desire in men are less well-known. Some men have issues with desire due to low testosterone levels. In these situations, testosterone replacement therapy has been shown to improve libido (118). Testosterone treatment in men with

Type 2 Diabetes and hypogonadism had improved libido as well as satisfaction, erectile function, and orgasm (51, 119).

Due to the finding that SSRIs result in delayed ejaculation (11), these medications have been used off-label for the treatment of premature ejaculation (11, 48, 49). Sex therapy and topic anesthetics have also been used to treat premature ejaculation (49).

Unfortunately, treatment options for delayed ejaculation usually focus on treating any underlying cause. Alpha 1 adrenergic agonists, alpha 2 adrenergic antagonists, and dopamine agonists have been explored as treatment options for delayed ejaculation (113).

Animal studies have found that cannabinoid receptor 1 agonist anandamide may reduce the ejaculatory threshold and improve delayed ejaculation (102).

1.4.2 Treatment for Women

Currently, there is only one drug on the market for female sexual dysfunction.

Flibanserin is a 5HT1A agonist/5HT2A antagonist that is available for women with

HSDD (17). Unfortunately, Flibanserin has a few side effects (67, 96). Bremelanotide, or

PT-141, was developed by the company Palatin, and showed promise for treating women with HSDD. Bremelanotide was an MC3 and 4R agonist that was shown to have positive effects on female animals (99). In clinical trials, this drug increased arousal in women, and increased satisfaction after sex (107). Unfortunately, the intranasal form of

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bremelanotide was found to have adverse effects on blood pressure, so clinical trials were discontinued (76, 124). New clinical trials investigated bremelanotide administered subcutaneously, with similar effects on sexual satisfaction and fewer side effects (24).

Mu-opioid receptor antagonists have been investigated in the treatment of FSAD with no benefit compared to placebo (94). Similarly, treatments that have been successfully used for male sexual dysfunction have had inconsistent results when tested in females (70,

110). However, intranasal oxytocin has shown improvement of both female sexual function and depression when administered long-term (89).

1.5 Animal Models of Sexual Dysfunction

To advance our understanding of sexual dysfunction, animal models have been used extensively to research the pathophysiology and potential treatments. In the 20th century, larger animals such as monkeys, dogs, and rabbits were popularly used to study erectile dysfunction (65). Rodents have become the model of choice for the ease of obtaining and maintaining the animals. Rodents display measurable sexual behaviors that are arguably analogous to human sexual behaviors. One group of researchers using electroencephalography data to investigate sexual function has found many similarities between the brains of humans and rats (57). For the male rodent, these behaviors begin with anogenital investigation of the female, which precedes multiple mounting attempts

(60, 98). The male will repeatedly mount the female, occasionally reaching intromission, or insertion of the penis, which can be identified by rhythmic deep thrusting. The male typically has to reach intromission several separate times before reaching ejaculation, which is characterized by the male freezing, falling off of the female, and subsequently losing interest in sexual activity. In rats, this post-ejaculatory refractory period can be

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followed by more ejaculation attempts in one night before becoming sexually exhausted

(98). Comparing these behaviors to human sexual behavior is up for debate, but Pfaus suggested that anogenital investigation in rodents falls under the same category as sexual desire in humans, mounting and intromission are reminiscent of copulation in humans, and finally ejaculation is seen in both rodents and humans (98). Rodents, especially rats, have also been used as models for erectile dysfunction by manipulating the cavernous nerve. To this end, the intracavernosal pressure of rats after stimulation of the cavernous nerve has been tested under various conditions (65). Radical prostatectomies have also been used to test erectile function, as these surgeries often lead to cavernous nerve injury

(65).

Female rodents use many behaviors to entice the male, which are believed to indicate a desire to engage in sexual behavior. These behaviors include solicitations, approaching males and then rapidly running away, hops and darts during the mounting period, and pacing the male by running away and returning (98). The primary measure of receptivity to copulation in rodents is lordosis, which is a reflexive posture defined by an arching of the back such that the head and hindquarters are raised above the central back

(98). Lordosis is often characterized using the lordosis quotient, which takes into account how often the lordosis posture is apparent when the male is attempting to mount (132).

Pfaus places lordosis into the same category as orgasm for humans (98). A model for hypoactive sexual desire disorder used two adjacent chambers housing a one male and one female, but only allowing the female to travel between compartments (117). In this way, the female could avoid the male or approach the male, with approaching behaviors representing sexual desire.

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1.6 Central Control of Sexual Function

Decades of research have gone into attempting to disentangle the complex interactions of central nervous system pathways underlying various aspects of sexual function. Through lesioning studies, tracing studies, pharmacological studies, and imaging studies, many of the neurotransmitters and brain regions involved have been identified, but the most of the pieces have not been connected. The desire to engage in sexual activity seems rooted in the dopaminergic and opioid systems, which control reward and pleasure (43, 101). Sensory systems are important for determining interest in a partner, particularly through the visual (130) and olfactory systems (43, 69, 135).

Erection has been found to involve the medial pre-optic area (MPOA), the paraventricular nucleus of the hypothalamus (PVN), the medial amygdala, and the spinal cord (27, 38, 47, 79). Dopamine, oxytocin, opioids, melanocortins, nitric oxide (NO), and norepinephrine have all been implicated in the central control of erection (2, 8, 27, 47).

The autonomic system plays a known role in erection (47). Parasympathetic innervations from the S2-S4 branches result in increased blood flow to the penis, leading to erection.

Because patients can still have erections with sacral lesions, but cannot have erections with a lesion at the T9 level or higher, it is thought that sympathetic inhibition is also required for tumescence (2). On the other hand, ejaculation is a spinal reflex, and can occur despite a complete transection of the spinal cord at T9 (18, 26) . Parasympathetic control is important for the emission portion of ejaculation, while sympathetic control drives expulsion (26). Brain regions do have an impact on ejaculation in humans as seen through PET imaging, including the midbrain, thalamic nuclei, and cerebellum (59).

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Female sexual behavior has implicated some overlapping brain regions and neurotransmitters with male sexual behavior. Estrogen receptor (ER), particularly alpha, has been found to be important for female sexual behavior in rodents (83, 106). Lordosis in animals requires the concerted effort of estrogen and progesterone. Because of this, when using ovariectomized females in studies, exogenous estrogen and progesterone must be administered to induce receptivity (132). Female sexual behavior studies have particularly implicated the ventromedial nucleus of the hypothalamus (VMH), the

MPOA, the periaqueductal gray (PAG), as well as the PVN (6, 28, 30, 39, 40, 78, 93).

Neurotransmitters found to affect lordosis when stimulated centrally include norepinephrine, acetylcholine, oxytocin, vasopressin, and serotonin (72). While most studies in female animals focus on lordosis, there are some studies that explore the role of neurotransmitters in sexual motivation. Apomorphine, a dopamine agonist, has been found to inhibit sexual desire in female rats (117).

1.7 The Melanocortin System and Sexual Dysfunction

Melanocortins are a group of peptide hormones with a wide range of actions throughout the body. They are produced by the cleavage of a Pro-opiomelanocortin

(POMC) polypeptide. POMC is produced by POMC neurons which are primarily in the arcuate nucleus (ARC) of the hypothalamus as well as the Nucleus Tractus Solitarius

(NTS) in the brainstem (25, 129). POMC is cleaved into multiple peptides that include adrenocorticotropic hormone (ACTH), β-endorphin as well as the α, β, and γ melanocyte stimulating hormones (MSH). These hormones act on five different melanocortin receptors, Melanocortin 1,2,3,4, and 5 Receptor (MC1-5R) (133). MC3 and 4R are primarily found in the central nervous system and have been implicated in the central

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control of sexual behavior (88). The melanocortin system received interest in sexual behavior research when it was found that melanocortin receptor agonists affected erection in male rodents (7) and receptivity in females (123).

In male mice, it has been observed that an intranasal administration of bremelanotide can lead to neuronal activity, as measured by c-fos, in the PVN and the supra-optic nucleus (SON) of the hypothalamus (87). Male mice that do not express melanocortin 4 receptors (MC4R) are reported to have erectile dysfunction and difficulty with ejaculation (126). Our own lab has found that reduced melanocortin production by insensitive POMC neurons reduces male sexual behavior, which may be associated with reduced MC4R expression in the hypothalamus (37). One downstream target of POMC is the PVN, which is thought to be important for regulating male sexual behavior (22), and may be mediated by melanocortins (112).

There is reason to suspect similar involvement of the melanocortin system in females. For example, after administration of α-MSH subcutaneously, there is an increase in female rat behaviors that are considered to be analogous to human feelings of sexual desire (99). An injection of a retrograde virus, pseudorabies virus, into the clitoris and vagina of a rat revealed that the PVN is included in the circuitry of female sexual behavior

(78, 127). Another study utilizing pseudorabies virus injected into the clitoris and vagina of a rat not only found labeling in the PVN but showed that it was associated with MC4R and, to a lesser extent, oxytocin (42). Bremelanotide has been shown to increase solicitation behavior in female rats (99). Injections of α-MSH into the VMH resulted in increased lordosis behavior in female rats, which was inhibited using an MC4R antagonist (28).

There is also evidence to support a role for MC4R in fertility, as MC4RKO mice have a

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decreased ovulation rate at an earlier age compared to controls (108). Between the age of

4 and 7 months, these mice are found to have altered ovarian histology as well as LH concentrations (23). Taken together, the evidence suggests that the melanocortin system is a strong candidate for a central target in the development of therapies for sexual dysfunction in both males and females.

1.8 Study Objectives

Not only is there strong evidence supporting the role of melanocortins and the

MC4R in regulating both male and female sexual dysfunction, but the clinical trials targeting these systems have had marginal success (24, 107). To make further advances in the treatment of sexual dysfunction, it would be useful to understand the pathways through which melanocortins are involved. This would allow for the identification of downstream targets that may have fewer side effects in patients. Such targets could also be useful to investigate as alternative therapies if melanocortin agonists are ineffective in certain patients. While certain nuclei in the brain are known to contain melanocortin receptors and are also implicated in the control of various types of sexual disorders, there is still a huge gap in our knowledge of the neurocircuitry connecting this information.

The overall goal of the current study was to elucidate a melanocortin pathway in the hypothalamus underlying male and female sexual behavior. Our primary target for these studies was the PVN. This nucleus contains MC4R and studies have shown its potential involvement in mediating sexual behavior. Furthermore, our lab has previously found that insensitive POMC neurons, which project to MC4 receptors in the PVN, lead to both inhibited sexual behavior and decreased MC4R expression (37). In these studies, we express MC4R exclusively in the PVN to test its role in mediating

15

sexual function. This work is significant because it provides evidence for a direct connection between the melanocortin and PVN, implicating both in the treatment of sexual dysfunction for both men and women, it carefully characterizes the sexual functions affected by the pathway, and it takes into account such factors as age and metabolism. Furthermore, we identify one population of neurons within the PVN and

SON, oxytocin neurons, that mediates this effect, providing support for the development of therapeutics that target the oxytocin system in treating sexual dysfunction.

The objectives of the study are summarized below:

1. To determine the role of melanocortin 4 receptor on Sim1 neurons in

male sexual behavior (Chapter 2).

a. Assessment of sexual function in MC4RKO mice compared to WT

controls

b. Assessment of sexual function in mice with MC4R expressed

exclusively on Sim1 neurons compared to MC4RKO and WT mice

c. Assessment of ICV administration of αMSH on sexual function across

genotypes

2. To determine the role of MC4R on oxytocin neurons in male sexual

behavior at a young age (Chapter 3).

a. Assessment of sexual function in two-month-old MC4RKO mice

compared to WT controls

16

b. Assessment of sexual function in two-month-old mice with MC4R

expressed exclusively on Sim1 neurons compared to MC4RKO and

WT mice

c. Assessment of sexual function in two-month-old mice with MC4R

expressed exclusively on oxytocin neurons compared to MC4RKO and

WT mice

d. Determination that metabolic phenotype associated with MC4R

manipulation is independent of sexual behavior changes

3. To determine the role of MC4R on oxytocin neurons in female sexual

behavior at a young age (Chapter 4).

a. Assessment of sexual function in two-month-old MC4RKO mice

compared to WT controls

b. Assessment of sexual function in two-month-old mice with MC4R

expressed exclusively on Sim1 neurons compared to MC4RKO and

WT mice

c. Assessment of sexual function in two-month-old mice with MC4R

expressed exclusively on oxytocin neurons compared to MC4RKO and

WT mice

d. Determination that metabolic phenotype associated with MC4R

manipulation is independent of sexual behavior changes

17

Chapter 2

Sim1 Neurons are Sufficient for MC4R-Mediated Sexual Function in Six-Month-Old Male Mice

*Manuscript to be submitted for publication

2.1 Introduction:

Sexual dysfunction affects a large number of men worldwide. Epidemiological statistics vary depending on the age of the population, the type of sexual dysfunction in question, and the comorbid factors considered (26). Most studies focus on erectile dysfunction, although men can also experience difficulties with interest, desire, ejaculation, and orgasm (15, 26). Between 9% and 54% of men across different countries have been reported to have experienced erectile dysfunction, with incidence increasing with age (22, 23, 28, 31). Ejaculation disorders, particularly premature ejaculation, are reported by 20-30% of men (21, 34).

Treatment options for men experiencing erectile dysfunction include phosphodiesterase 5 inhibitors, such as Viagra, injection of the penile tissue with prostaglandin E1 or similar drugs, and surgical prostheses (16, 30). Phosophodiesterase 5 inhibitors improve erectile function in a significant number of men, but in some cases, particularly those with underlying conditions such as diabetes, these medications are 18

ineffective (7, 10). For these patients, it is important to consider central mechanisms as therapeutic targets for sexual dysfunction. For premature ejaculation, selective serotonin reuptake inhibitors and local anesthetics have been used for cases that do not seem to have a secondary, treatable cause (16). Another ejaculation disorder, delayed ejaculation, is less commonly reported and lacks effective treatments (1).

The melanocortin system is a promising central target for the treatment of sexual dysfunction in men. One study found that a melanocortin 3/4 receptor agonist, bremelanotide, increased satisfaction levels in men who were also taking Viagra (36).

Bremelanotide has also been found to increase blood pressure and decrease heart rate

(47). A melanocyte stimulating hormone (MSH) analog, melanotan-II (MT-II), has been found to induce erections in men, but it has a high incidence of unwanted side effects such as nausea and excessive yawning (45, 46). The results of these studies confirm the importance of understanding which central pathways mediate the effects of melanocortins, not only to develop treatments for sexual dysfunction, but also to minimize side effects of pharmacotherapy.

Mouse models offer an excellent avenue for understanding the mechanism underlying melanocortin-driven sexual behavior and function. For example, mice lacking the melanocortin 4 receptor (MC4R) are reported to show reduced sexual motivation as well as reduced ejaculation efficiency (42). To better understand the interaction between the central nervous system and sexual function, our lab has investigated the involvement of the pro-opiomelanocortin (POMC) system. Mice bred to have POMC neurons that were insensitive to circulating insulin and leptin were found to show decreased mounting behavior, indicating reduced sexual motivation (11). These mice also showed reduced α-

19

MSH production and reduced expression of MC4R. Despite rodent and human studies implicating the melanocortins in sexual behavior, the brain nuclei containing the critical melanocortin receptors are unknown.

The MC4R, primarily found in the brain, is known to be located in key hypothalamic nuclei downstream of POMC neurons, such as the paraventricular nucleus of the hypothalamus (PVN) (40). Balthasar and colleagues previously generated a mouse model in which MC4R is expressed in specific tissues that express Single-minded homolog 1 (Sim1) (5, 37). Sim1 is a transcription factor that, when knocked out, results in a lack of development of the PVN and supraoptic nucleus (SON) (27). Sim1 is highly expressed in adult PVN neurons as well (41), allowing for the utilization of Sim1 as a target for this region. Using the cre-lox system to specifically express MC4R only on

Sim1-cre neurons, it was shown that Sim1 MC4Rs are involved in satiety. MC4R null mice become obese over time, while expressing MC4R solely on Sim1 neurons attenuated this phenotype (5). Further studies have found evidence that this effect may be mediated by glutamate (37, 48).

In the current study, we utilize this mouse model as a tool to explore the neurocircuitry of melanocortin 4 receptor-mediated sexual behavior. Specifically, we test the specific involvement of Sim1-cre melanocortin 4 receptors in sexual performance parameters of male mice. Ultimately, understanding the mechanisms underlying melanocortin-mediated sexual behavior may pave the way for future treatments of male sexual dysfunction.

2.2 Methods:

Animal production and care

20

MC4RKO mice, a previously established mouse model, were purchased from The

Jackson Laboratory (loxTB Mc4r, 006414). The transcription blocker preventing expression of MC4R in these mice is flanked by loxp sites, such that the presence of Cre recombinase will result in the removal of the transcription blocker and subsequent expression of MC4R in tissue-specific sites. Generation of tbMC4Rsim1 mice was accomplished by breeding Sim1-cre mice (The Jackson Laboratory, 006395) with mice that were heterozygous for the MC4R null allele. Experimental mice were bred to be heterozygous for Sim1-cre but homozygous for the MC4R null allele. Control mice included wild-type (WT) littermates as well as mice that were only heterozygous for

Sim1-cre but had normal expression of the MC4R gene. Genotyping was confirmed by sending tissue to Transnetyx, Inc. for testing by real-time PCR.

Mice were housed in the University of Toledo College of Medicine DLAR facilities where they were given ad libidum food and water on a 12:12 light dark cycle.

Food was standard rodent chow. All procedures were reviewed and approved by the

University of Toledo College of Medicine Animal Care and Use Committee.

Sexual Behavior

Prior to six months of age, males were exposed to primed, ovariectomized female mice three separate times in order to gain sexual experience. To prime the females, a subcutaneous 100µL dose of β-estradiol-3-benzoate in sesame oil (200µg/mL) was given

48 hours prior to pairing, and then an intraperitoneal 125 µL dose of progesterone

(4mg/mL) was given 7 hours prior to pairing (13, 35). Pairing was done with sexually experienced female mice from 8pm-9am, during their normal period of activity.

After male mice reached 6 months of age, pairing was videotaped (DVR Swann

21

4500 and T850 Day and Night Security Camera security system) and analyzed for sexual behaviors during 8pm-2am. This timespan was determined to include the majority of relevant sexual behaviors. Sexual behaviors measured included anogenital sniffing, mounting, intromission, and ejaculation. Motivational behaviors included length of time spent in the anogenital sniffing phase, latency to mount, and mounting behavior within the first twenty minutes of copulation. Latency to mount within twenty minutes was assigned a value of twenty minutes if mounting was not initiated until after that time. Mounting was defined as placing two paws on the back of the female and attempting to thrust.

Intromission was defined as deep, successful thrusts. The ratio of successful intromission to mounting attempts was determined to be a measure of intromission efficiency.

Ejaculation was defined as the moment during intromission when males freeze, fall over, and subsequently lose interest in the female.

Cannulation

A guide cannula (PlasticsOne, 2.3mm) was surgically implanted into the lateral ventricle using a stereotaxic apparatus. Mice were anesthetized using a ketamine/xylazine mixture and then the cannula was implanted using the following coordinates: AP: -0.22

ML: +1.13 DV: -1.95. Mice were given three days to recover, were singly housed, and then were tested for a stretching, yawning, and grooming reflex in response to αMSH administration as previously described (3, 4). Administration of αMSH or saline was done using a 5µL Hamilton syringe attached with microrenathane tubing (BrainTree Scientific) to an internal cannula (PlasticsOne, 1mm). The stretching, yawning, and grooming reflex testing was done between 10am and 2pm. One µL of 3µg/µL aMSH or 1µL of 0.9% saline was administered into the lateral ventricle and mice were returned to their home cage. Ten

22

minutes following injection, mice were observed for two hours and grooming, stretching, and yawning behaviors were noted in 15s intervals.

Sexual behavior was also tested immediately following administration of αMSH.

Prior to being paired with an experienced partner, mice were given an ICV injection of either 1µL of 1µg/µL αMSH or 1µL of 0.9% saline using a Hamilton syringe. All mice were tested in both conditions with three days between behavioral testing, in randomized order.

Statistics

GraphPad Prism was used for all statistical analysis. All data in figures are represented as mean ± SEM. One-way ANOVA was used to compare more than two groups followed by Fisher’s LSD post-tests. For experiments that tested the same mice in both a saline and αMSH condition, a two-way ANOVA was used, followed by Fisher’s

LSD post-tests to compare within conditions. Statistical significance was defined as p<0.05. In figure legends, *, p<0.05; **, p<0.01; ***p<0.001, ****p<0.0001.

2.3 Results:

MC4R null mice have impaired sexual function

Sexual function was assessed in 6-month-old male MC4RKO mice by pairing them with hormonally-primed ovariectomized females. These mice exhibited the expected phenotype of increased weight gain (p<0.0001), which has been well established in the literature (37) (Fig. 1A). Males showed no difference in mounting attempts, latency to mount, or number of times intromission was reached (Fig. 1B-D).

However, mice were found to have a trend towards decreased intromission efficiency indicating difficulty with erectile function (p=0.0506) (Fig. 1E). Intromission efficiency

23

was defined as the percentage of times intromission was reached during mounting attempts. The ability of the males to reach intromission suggests that they were able to achieve erection. However, because these mice required more mounting attempts to reach intromission than controls, these mice may have had difficulty maintaining sufficient penile erection. MC4R null mice also had a complete inability to achieve ejaculation

(p=0.0009) compared to WT mice (Fig. 1F). These results indicate that MC4RKO mice had a significantly impaired ability to ejaculate and showed some signs of erectile dysfunction.

MC4R on Sim1 neurons are important for sexual function

Due to the dense expression of MC4R in the paraventricular nucleus, it was hypothesized that this nucleus may contain MC4R relevant for sexual behavior. To test this, mice were generated to express MC4R solely on Sim1 neurons, which are found predominantly in the paraventricular nucleus. We confirmed previous studies that found that these mice also have a phenotype of weight gain compared to WT (p=0.0001) and

Sim1-cre (p=0.0013) control mice (Fig. 1A). However, no weight difference was seen compared to MC4RKO mice.

Sexual dysfunction was assessed in male tbMC4Rsim1 mice and then compared to both MC4R null mice and controls. The number of mounting attempts was not different between any groups (Fig. 1B). No significant difference was found between WT controls and Sim1-cre controls for weight (p=0.6099), mounting (p=0.3402) or any other parameter, so for ease of interpretation, WT controls were used to represent all control mice for the figures. Unlike MC4R null mice, tbMC4Rsim1 mice showed normal sexual parameters (Fig. 1B-F). Intromission efficiency was significantly different across groups

24

(F(2,25)=4.472, p=0.0219), with a significant increase in efficiency in tbMC4Rsim1 mice compared to MC4RKO (p=0.0073) (Fig. 1E). There was also a significant difference across genotypes in the percentage to reach ejaculation (F(2,25)=7.331, p=0.0031), due to a recovery of the ability to ejaculate in tbMC4Rsim1 mice compared to MC4RKO mice

(p=0.0041) (Fig. 1F). This suggests that MC4R on Sim1 neurons are sufficient to mediate the effects of melanocortins on erectile function and ejaculation.

Willingness to mount, as measured by behaviors such as the latency to mount and mounting attempts in the initial pairing period, have been shown to indicate the sexual motivation of the male and has been considered analogous to sexual desire in humans

(32). One method of assessing sexual motivation involves measuring a male’s interest in a female in the first 10 or 20 minutes after pairing (11). Therefore, sexual behavior in the first 20 minutes of pairing was analyzed. Although MC4RKO mice exhibited no differences in number of mounts within the initial twenty minutes (Fig. 2A), their latency to mount was significantly increased (p=0.0472) (Fig. 2B). This finding is consistent with previous studies that examined the effect of MC4R on sexual behavior in mice (42). In tbMC4Rsim1 mice, sexual motivation did not differ statistically from controls (Fig. 2A-B), suggesting that the motivational defects seen in the MC4RKO are mediated by Sim-1 neurons.

Exogenous αMSH affects grooming behavior through Sim1 neurons

Exogenous αMSH has been reported to induce a stretching-yawning-grooming response, which is also accompanied by erection (4, 29). Thus, we investigated whether

ICV administration of αMSH works through PVN MC4R to elicit these behavioral responses. αMSH did significantly increase grooming (p=0.0002), stretching (p=0.0003),

25

and yawning (p=0.0172) behaviors in control animals (Fig. 3A-C). We found that

MC4RKO mice did not have increased grooming, stretching, or yawning in response to

αMSH administration as expected, but only grooming was found to be induced by αMSH in tbMC4Rsim1 mice (p=0.0411) (Fig. 3A). There was a significant interaction (F(2,

54)=6.293, p=0.0035), significant effect of genotype (F(2, 54)=4.419, p=0.0167), and significant effect of treatment (F(1,54)=8.861, p=0.0044) for grooming behaviors. A two- way ANOVA on stretching behavior also revealed a significant interaction (F(2, 54)=

3.622, p=0.0334) with a main effect of genotype (F(2, 54)= 6.637, p=0.0026) and treatment (F(1, 54)=9.000, p=0.0041). The main effect of genotype shows that WT mice stretched more often than either MC4RKO (p=0.0009) or tbMC4Rsim1 mice (p=0.0126).

There was no significant interaction or main effects on yawning behavior. These data suggest that only the grooming response to central αMSH administration is mediated by

MC4R on Sim1 neurons.

Exogenous αMSH prevented ejaculation in tbMC4Rsim1 mice

To assess the effect of exogenous αMSH on sexual function, sexual behavior was tested after the ICV administration of 1µg of αMSH. Genotype affected initial mounting behavior (F(2,48)=3.372, p=0.0426) with MC4RKO mice showing reduced mounting compared to WT (p=0.0143) (Fig. 4A). Latency to mount was also significantly affected by genotype (F(2,48)=10.03, p=0.0002), with MC4RKO (p<0.0001) and tbMC4Rsim1

(p=0.0081) mice taking longer than WT mice (Fig. 4B). αMSH had no noticeable effect on sexual motivation compared to saline in the first twenty minutes of sexual behavior.

These findings indicate that sexual motivation is not impacted by ICV αMSH. We then extended our observations to cover a six-hour time period to allow an opportunity for

26

successful intromission and ejaculation. Contrary to our hypothesis, tbMC4Rsim1 mice had worse erectile function (p=0.0118) when administered αMSH compared to saline (Fig.

4C). A main effect of genotype (F(2,50)=4.684, p=0.0137) also revealed that MC4RKO mice had a lower intromission:mount ratio compared to both WT (p=0.0055) and tbMC4Rsim1 mice (p=0.0243). There was a main effect of genotype on the percentage of mice to reach ejaculation (F(2,50)=10.06, p=0.0002), with a higher percentage of wild- type mice successfully ejaculating compared to both tbMC4Rsim1 (p=0.0158) and

MC4RKO mice (p<0.0001) αMSH administration also resulted in an inability for tbMC4Rsim1 mice to reach ejaculation (p=0.0026) (Fig. 4D). Interestingly, αMSH had no effect on the sexual function of WT mice.

2.4 Discussion:

In this study, we have found that MC4R null mice have a complete inability to reach ejaculation as well as decreased intromission efficiency. These parameters were restored by expressing MC4R only on Sim1 neurons, indicating that these receptors in

Sim1 neurons are sufficient to permit normal erectile function and ejaculation. This is the first study to demonstrate that direct melanocortin action on Sim1 target neurons is necessary for sexual function. Taking into account our previous studies in mice with leptin and insulin-insensitive POMC neurons (11), this study provides evidence that

MC4R-expressing Sim1 neurons receive input from arcuate POMC neurons and together form part of a neural circuit underlying male sexual function (Fig. 5).

We tested several aspects of the sexual response. Sexual motivation, the human analogue of sexual desire, was investigated via pre-copulatory behaviors (anogenital sniffing) and willingness to engage in mounting behavior (latency to mount and initial

27

mounting attempts) (32). We saw an increase in latency to mount was found in MC4RKO mice, but no other difference in sexual motivation. In contrast, Van der Ploeg and others

(11, 42) found a clear lack of sexual motivation in MC4R null mice that was not seen in our studies. In contrast to that report, we ensured that all mice in our study had previous copulatory experience prior to testing. This step may have eliminated delays in engaging in sexual activity due to inexperience and minimized differences between groups.

Measuring erectile function in mice during copulation is complicated, as rigidity cannot be directly measured in that setting. Instead we used successful intromission as a proxy for this measure. Specifically, we examined intromission behavior and the intromission to mounting ratio. We found that the ratio of intromission to mounting was significantly decreased in MC4RKO mice and recovered in tbMC4Rsim1 mice.

In our copulation studies, we hypothesized that αMSH would result in improved erectile function in control mice and tbMC4Rsim1 mice. Surprisingly, αMSH had no effect on control animals. This finding may indicate that the normal copulatory function of control mice could not be further improved by αMSH due to a physiological ceiling.

Alternatively, improvement may be possible in control animals under other experimental paradigms; specifically, previous pharmacological studies primarily investigated non- contact erection, while we measured copulatory function. Our pharmacological experiment adds to wealth of complex data regarding the effect of centrally administered

αMSH on copulatory function.

Finally, ability to ejaculate was examined by the percent of mice able to achieve ejaculation. We found that MC4RKO mice were unable to reach ejaculation and tbMC4Rsim1 mice exhibited normal ejaculation function. These findings are consistent

28

with a previous study showing reduced ejaculation efficiency in MC4R null mice (42).

The role of the PVN is further supported by findings of reduced intromission efficiency and increased ejaculation latencies in rats that had lesions in their PVN (24). However,

ICV αMSH resulted in a decrease in erectile function and ejaculation in tbMC4Rsim1 mice. This is contrary to what was expected based on our results that suggest MC4R in the PVN permit normal sexual behavior. One potential explanation lies in the dosage of

αMSH. Whereas endogenous levels of MC4R ligands permitted normal sexual behavior in tbMC4Rsim1 mice, it is possible that the exogenous dosage of αMSH used was inappropriate. Due to the actions of αMSH on excessive grooming behavior with administration of 3µL, a lower dosage (1µL) was selected for sexual behavior testing to reduce the interference of αMSH-induced grooming (4). Other considerations include the non-specificity of αMSH, especially given that the ICV injection is not specific to the

PVN. One potential explanation for these findings lies in the role of MC4Rs and MC3Rs in the brain. While MC4R is typically thought to have a stimulatory effect on erectile function, MC3R has been hypothesized to be inhibitory (18). Because αMSH acts on both receptors, it is possible that MC4R only on Sim1 neurons do not have a strong enough stimulatory effect to balance out an inhibitory MC3R effect elicited by exogenous αMSH.

Given the sufficiency of Sim1 MC4R expression to permit efficient intromission and ejaculation with endogenous melanocortin release, these results emphasize the fact that pharmacological treatment does not replicate the normal function of this circuitry. Further studies are necessary to better understand the relationship between these receptors.

In mice as in humans, ejaculation is a clear, measurable response. Ejaculation is heavily controlled by autonomic and motor neurons through the spinal cord. Lesioning

29

studies and tracing studies in animals have implicated regions of the brain such as the medial preoptic area (MPOA) as well as the PVN in the central control of ejaculation (2).

One study utilizing positron emission tomography to examine brain activity in humans during ejaculation found activity in many mesodiencephalic regions, but not in the hypothalamus (17). Although it has been suggested that rodents may experience orgasm- like responses (33), we were unable to measure orgasm in our mice. Interestingly, it has been suggested that the loss of interest felt following ejaculation is similar to the sensation of satiety felt after eating (14), a state in which the melanocortin 4 receptor is known to be involved.

Studies have found that administration of MC4R agonists into the lateral ventricles of the brain of rodents results in erection, along with yawning, stretching, and grooming (4, 18). We hypothesized that ICV administration of αMSH would increase these melanocortin-mediated behaviors in all mice except MC4R null mice. While we did see increases in grooming and stretching in control animals, only grooming was increased in tbMC4Rsim1 mice. This effect may indicate that MC4Rs in Sim1 neurons regulate grooming, but not stretching and yawning. This result is supported by findings that

αMSH in the paraventricular nucleus elicits a grooming response (8, 44). Interestingly, one study noted that these αMSH-induced behaviors only occur in the presence of stressful handling procedures. These authors suggest that αMSH maintains grooming but does not initiate it, so these behaviors might not be present in the absence of stress (43).

Nevertheless, understanding the neurocircuitry underlying these behaviors may permit targeted drug development for erectile dysfunction without such unwanted side effects.

30

MC4R on Sim1 neurons also play a role in regulating eating behavior (5, 37, 48).

Obesity is thought to contribute to sexual dysfunction in men (9). As seen in previous studies, the MC4RKO mice did become considerably obese (5, 37). Our tbMC4Rsim1 mice were also obese, which is supported by the literature, although at this age we did not see the reportedly attenuated weight gain compared to MC4R null mice (5, 37, 48). It is unknown how obesity impacted the results of these studies, although it should be noted that tbMC4Rsim1 mice were still able to mate as well as controls despite marked obesity.

Additional studies will be required to disentangle the effects of obesity and melanocortin deficiency.

More investigation is necessary to determine which Sim1 neurons underlie the restoration of erectile function and ejaculation in tbMC4Rsim1 mice. The MC4R, expressed primarily in the brain, is found throughout the hypothalamus (38), hippocampus, cortex, and amygdala (40). It is also found in the spinal cord (40), pituitary, pelvic ganglion, and penis (42). Sim1 neurons are found in the paraventricular nucleus of the hypothalamus and the amygdala (19), making these likely candidates for the location of the neuronal population involved in regulating sexual behavior. Indeed, fMRI studies in humans have also implicated the hypothalamus and amygdala in regulating erection (12). While the amygdala should also be investigated for its potential role in melanocortin-dependent sexual function, several lines of evidence support our proposed interpretation that the PVN is likely to play a role in the observed effects (Fig.

5). A tracing study that injected pseudorabies virus into the corpus cavernosus of rats found virus in the paraventricular nucleus (25). Furthermore, a study in rats found that lesioned parvocellular and magnocellular neurons in the paraventricular nucleus resulted

31

in delayed ejaculation and reduced intromission ratios (24), supporting a role for Sim1 neurons in sexual function. Paraventricular nucleus projections to serotonergic neurons in the medulla have been found to be involved in regulating male sexual function (6). Sim1 expressing cells in that nucleus include oxytocin, TRH, CRH, vasopressin, and somatostatin neurons (20); both oxytocin and serotonin neuronal projections have been found on preganglionic sacral neurons that control penile function (39). Narrowing down the relevant neurocircuitry may assist in the development of drugs that can target sexual function without impacting any other melanocortin pathways.

In conclusion, we have shown that MC4R signaling in Sim1 neurons regulate erectile function and ejaculation in male mice at 6 months of age. These receptors also seem to mediate grooming behavior, but not stretching or yawning. These results and future investigation of implicated melanocortin neurocircuitry may allow for the development of more specific therapeutic targets for improving sexual function. In addition, determining how these melanocortin circuits diverge from those regulating energy balance and metabolism may permit the development of weight loss medication free of sexual side-effects.

32

2.5 Figures:

W e ig h t (g ) A B M o u n tin g A tte m p ts 6 0 W T 8 0 * * * * * * * M C 4 R K O s im 1 6 0 4 0 tb M C 4 R 4 0 2 0 2 0

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o 0 .2 M 0 .0 0 .0 S i m 1 W T M C 4 R K O tb M C 4 R Figure 1. Sexual behavior in MC4RKO and tbMC4Rsim1 mice. MC4RKO mice had an altered sexual behavior phenotype whereas mice expressing MC4R only on Sim1 neurons (tbMC4Rsim1) resulted in a sexual behavior phenotype consistent with WT mice. (A) Weight gain was significantly different across groups. Post-tests revealed MC4RKO and tbMC4Rsim1 mice had significant weight gain compared to WT controls (*) (N=9-12). (B) Mounting attempts between wild-type, MC4RKO mice, and tbMC4Rsim1 (N=8-10) was not significantly different. There was also no significance between groups with latency to mount (C) or intromission number (D). There was, interestingly, a significant difference between MC4RKO and tbMC4Rsim1 mice in intromission efficiency. (E) A trend (p=0.0506) towards MC4RKO mice being lower than WT was seen. (F) Percentage to reach ejaculation was significantly higher in both WT and tbMC4Rsim1 mice than MC4RKO mice.

33

A M o u n tin g A tte m p ts in 2 0 M in u te s L a te n c y to M o u n t in 2 0 M in u te s B 2 0 1 0 0 0 *

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34

A G r o o m in g B S tr e tc h e s 1 0 0 * * * 6 * * * S a lin e 8 0  M S H

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35

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R

M % 0 .0 0 .0 s im 1 s im 1 W T M C 4 R K O tb M C 4 R W T M C 4 R K O tb M C 4 R

Figure 4. ICV aMSH had an effect on tbMC4Rsim1 behavior (N=8-10). (A) A two-way ANOVA revealed no effect of αMSH treatment on number of mounts reached in the first twenty minutes across genotypes. (B) Similarly, there was no effect of treatment on latency to mount in the first twenty minutes. (C) αMSH reduced intromission efficiency in the tbMC4Rsim1 group. (D) In the αMSH treatment condition, tbMC4Rsim1 had significant impairment in reaching ejaculation within six hours compared to the saline condition.

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Sim1 PVN MC4R Neu Neu Sexual Behavior 3V

ARC POMC

Figure 5. Schematic of interpretation of results. POMC neurons in the arcuate nucleus (ARC) project to the PVN onto Sim1 neurons that express MC4R, which regulates downstream sexual behavior.

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Chapter 3

Hypothalamic Melanocortin 4 Receptors Influence Male Sexual Behavior in Young Mice

*Manuscript intended for publication

3.1 Introduction:

Sexual dysfunction has a profound effect on men throughout the world. At least one fifth of men experience some form of sexual dysfunction during their lifetime (23,

27). Although treatments such as phosphodiesterase inhibitors exist for erectile dysfunction, there is a lack of research supporting medication for other forms of sexual dysfunction (29). Central mechanisms have become a target for addressing these issues.

Selective Serotonin Reuptake Inhibitors that increase extracellular serotonin have been used as a treatment for premature ejaculation (23). The dopamine agonist, apomorphine has been explored as a treatment for sexual motivation and erectile dysfunction (20).

Drugs that target the melanocortin system have also been tested as a treatment for erectile dysfunction (36, 51). Increasing our understanding of the neurocircuitry underlying different sexual behaviors may lead to more focused treatment options.

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Many studies have investigated the involvement of the melanocortin system in regulating sexual behavior. Exogenous αMSH administration has been found to lead to erection in rodents (4). MC4R null mice have shown decreased levels of motivation for sexual activity, as well as reduced ejaculation efficiency (48). Our own studies of the melanocortin system, using mice with pro-opiomelanocortin (POMC) neurons that were insensitive to leptin and insulin, showed decreased motivation at 4 to 6 months of age, when these mice are also showing increases in adiposity and insulin insensitivity (14).

These mice had reduced expression of MC4Rs, suggesting that these receptors may be mediating sexual motivation downstream of POMC neurons. One hypothalamic nuclei that POMC neurons project to is the paraventricular nucleus (PVN) (45).

Our lab previously attempted to explore the mechanisms underlying sexual dysfunction in 6-month-old male mice by genetically expressing MC4R in Sim1 neurons.

Sim1 neurons have been used by multiple investigators as a tool for targeting paraventricular neurons, although they are also found in the supraoptic nucleus (SON) and amygdala (6, 31, 47). Our lab’s studies implicated Sim1 neurons in the neuronal pathway involved in regulating both ejaculation and intromission efficiency, however,

MC4R on Sim1 neurons also play a role in regulating eating behavior (6, 39, 53). At 6 months of age, these mice weigh twice the amount of control mice. It is therefore difficult to determine whether MC4R on Sim1 neurons directly regulate sexual function, or if sexual function is indirectly affected by weight.

One reason for investigating the paraventricular nucleus is that this nucleus, along with the supraoptic nucleus, contains oxytocin neurons. Oxytocin neurons comprise of a large portion of the population of Sim1 neurons (25), and are found in the PVN and SON

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(7), downstream of POMC neurons. Oxytocin neurons have been heavily implicated in mediating sexual behavior in both animals (3, 30, 42) and humans (8, 10), and it has even been hypothesized that these neurons may be mediated by MC4 Receptors (18, 33), although no direct connection has been made.

It has been well-documented that sexual function in men worsens with age (17,

22, 29, 32, 35). It is not entirely known why age negatively affects sexual function, but it is hypothesized that increased obesity and cardiovascular complications may play a role.

This is particularly high in men with comorbid factors, such as diabetes (46). Many animal studies do not take into account how age may be influencing their studies.

Previous mouse models used to study sexual dysfunction were between 3 and 6 months of age, when MC4R null mice have a clear obese phenotype that may be a confounding factor (11, 24, 41).

We hypothesize that oxytocin neurons may mediate MC4R-driven sexual behavior in male mice. Our present studies, using two-month old mice in order to reduce the potential confound of age-related obesity, show that melanocortin 4 receptors are involved in male ejaculation, which is recovered when these receptors are selectively expressed on Sim1 neurons and on oxytocin neurons. Furthermore, we provide evidence that this effect is independent of the weight gain caused by a lack of melanocortin 4 receptors. This study provides support for a central mechanism underlying ejaculation.

3.2 Methods:

Animal Production and Care:

All mice used in this study were kept in accordance with the University’s IACUC guidelines. MC4R knock-out mice were purchased from The Jackson Laboratory (loxTB

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Mc4r, 006414) and bred using heterozygous mice. A transcription blocker in the promoter for the MC4R gene prevents MC4R expression. This transcription blocker is flanked by loxp sites, such that cre recombinase-expressing tissue would result in the removal of the transcription blocker, and thus the expression of MC4R. Therefore, mice expressing cre on Sim1 neurons, and homozygous for the floxed MC4R gene would express MC4R only on Sim1-expressing neurons (tbMC4Rsim1). To generate tbMC4Rsim1 mice, a Sim1-cre-expressing mouse line (The Jackson Laboratory, 006395) was used to breed with the loxTB MC4R mice. Similarly, oxytocin neurons that expressed cre were used to generate mice that expressed MC4R only on those neurons (tbMC4Roxt). Mice were bred such that cre-expression was heterozygous, which has been shown to be sufficient in the past and would limit the chance for an unintended cre-specific phenotype. Genetics were confirmed by sending a piece of tail to Transnetyx, Inc. for automated genotyping using real-time PCR.

Mice were housed in DLAR facilities where they were given ad libidum water on a 12:12 light dark cycle. Food was given ad libidum.

Sexual Behavior Studies:

Male mice at the age of 7-8 weeks were exposed to primed, ovariectomized female mice three separate times to gain sexual experience. To prime the females, a subcutaneous 100µL dose of β-estradiol-3-benzoate in sesame oil (200µg/mL) 48 hours prior to sexual behavior, and then an intraperitoneal 125 µL dose of progesterone

(4mg/mL) 7 hours prior to pairing to prime females (16). Pairing was done with experienced male mice from 8pm-9am, during their normal period of activity. The fourth pairing was videotaped (DVR Swann 4500 and T850 Day and Night Security Camera

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security system) and analyzed for sexual behaviors during 8pm-2am, which was determined to include the majority of relevant sexual behaviors. Sexual behaviors measured included anogenital sniffing, mounting, intromission, and ejaculation.

Anogenital sniffing was defined as the period in which the male was sniffing the female’s genitals prior to the first mounting attempt. Mounting attempts were defined as any time the male places his forepaws onto the female’s back and attempted to thrust. Successful intromission was defined as deep, slow rhythmic thrusting during a mounting attempt.

The intromission to mount ratio was defined as the number of times intromission was reached per total mounting attempts. Ejaculation was defined as the male freezing and falling over following intromission.

Metabolic Studies:

MC4R null mice have a well-established phenotype in the literature as obese mice

(11, 24, 41). The early age of sexual behavior was determined because our lab has discovered that, due to the metabolic profile of MC4R null mice, after reaching adulthood they become too obese to successfully engage in the sexual act. Glucose tolerance test

(GTT), nuclear magnetic resonance (NMR) for fat content, and metabolic cages to assess energy expenditure were used to assess the metabolic profile of these mice. The same mice used for sexual behavior testing were used for all tests except a separate cohort was needed for the metabolic cages.

The morning after sexual behavior testing, Nuclear Magnetic Resonance (NMR) is used to assess fat content of the mice (BrukerOptics). At 9-12 weeks old, a glucose tolerance test (GTT) is done. The morning of the GTT, mice were fasted for six hours on alpha-dri bedding (starting at 8am). At 2pm, baseline glucose levels are obtained by

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measuring tail-vein blood. A 2g/kg dosage of dextrose was administered intraperitoneally and subsequent blood glucose levels were measured at 15, 30, 45, 60, 90, and 120 minutes post-injection using a glucometer for pets (AlphaTRAK 2, ADW Diabetes).

Mice were placed in metabolic cages (Columbus Instruments’ Comprehensive Lab

Animal Monitoring System) between 2 and 3 months of age in order to assess food intake, water intake, physical activity, and energy expenditure. Mice were allowed to acclimate to the cages for over 24 hours before data was collected. Data was collected every 20 minutes for each mouse for 72 hours before returning to their home cage

(Oxymax for Windows).

Hormone Assays:

Submandibular blood samples are taken at week 10-13. Serum was obtained by placing samples in the centrifuge for 10 minutes at 4°C at 4472 rcf and then collecting the clear fluid layer. Analysis was done using Testosterone and Estradiol ELISA kits

(CalBiotech). LH/FSH was measured by UVA using a multiplex assay.

Diet Induced Obesity:

For DIO tests, wild-type littermates were given HFD (OpenSource Diets, 60% fat content) instead of standard chow at 5 weeks of age. Mice were weighed weekly until their weights were comparable to MC4RKO mice at the time of their sexual behavior testing. DIO mice went through all tests as previously described.

Immunohistochemistry:

Upon euthanasia, mice were perfused and the brain was obtained. Testes of the males were weighed. The brain was sectioned in 35-40µm slices and then stored in cryoprotectant until immunohistochemistry was performed in order to identify the

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location of MC4R in the hypothalamus and its co-localization with Sim1 and oxytocin.

To verify that there are MC4R on Sim1 neurons in the PVN, brain sections were labeled overnight with rabbit anti-MC4R (Abcam ab24233). Td-tomato was used as a cre- reporter and the fluorescence was visible via confocal without the aid of an additional antibody. MC4R was visualized using an Alexa Fluor 488 secondary antibody (Donkey anti-rabbit IgG Alexa Fluor 488, Invitrogen, A21206). To verify that there are MC4R on oxytocin neurons in the PVN, brain sections were labeled overnight with rabbit anti-

MC4R (Abcam ab24233) and chicken anti-GFP (Aves Labs Inc, IgY0511FP12), because the oxytocin-cre expression was paired with GFP. Sections were exposed to Alexa Fluor

488 and 594–conjugated secondary antisera (donkey anti rabbit Alexa Fluor 594,

A21207, Life Technologies; goat anti chicken Alexa Fluor 388, A11039, life technologies) and observed using a confocal microscope (Leica).

Data Analysis:

All data in figures are represented as mean ± SEM. Parametric, two-tailed unpaired t-tests were used to compare two groups, and a one-way ANOVA was used to compare more than two groups followed by Fisher’s LSD post-tests. Correlation tests were used to determine the relationship between two continuous variables with results reported as R2 values. Statistical significance was defined as p<0.05. In figure legends, *, p<0.05; **, p<0.01; ***p<0.001; ****p<0.0001.

3.3 Results:

MC4R null mice have an altered sexual phenotype at two months of age

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To confirm the role of MC4R in sexual behavior, male MC4R null mice were paired with females at two months of age and filmed for six hours. Compared to wild type controls, MC4RKO mice had significantly increased mounting attempts (Fig. 1A; p=0.0016), but no difference in latency to mount (Fig. 1B; p=0.275). MC4RKO mice also reached intromission more times than WT mice (Fig. 1C; p=0.0135). The intromission:mount ratio, which is an indication of intromission efficiency and erectile function was not different between groups (Fig. 1D; p=0.3346), likely due an equal increase in both mounting and intromission. There was also an increased latency to ejaculation in the MC4RKO mice (Fig.1E; p=0.0015). Because increased mounting attempts and intromission was an unexpected finding, we considered the possibility that this increase was due to the increased latency to ejaculation. Mice typically do not continue to mount after reaching ejaculation, so if the wild-type mice were achieving ejaculation more successfully, they may not have needed as many mounting attempts as

MC4RKO. To visualize the potential relationship between latency to ejaculate and mounting attempts, we ran a correlation between the two measures and found a very strong positive correlation (Fig. 1F; r=0.9387, p<0.0001, R2=0.8812). This implies that the mice that took longer to ejaculate were the same mice that had higher mounting attempts.

Previous literature, including that from our own lab used the first twenty minutes of copulatory behavior to assess the interest level of the mice (14). Anogenital sniffing duration, or the period of time in which the male investigates the genitals of the female prior to mounting, was not significantly different between MC4RKO and WT mice (Fig.

2A; p=0.1116). Interestingly, the MC4RKO mice had significantly increased mounting

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attempts even in the first twenty minutes compared to controls (Fig. 2B; p=0.0494), however, if the mice who achieved ejaculation (Fig. 2C; p=0.1226) within the first twenty minutes are removed, the significance is lost (Fig. 2D; p=0.4368). This suggests that while the data appear to indicate that MC4RKO are more interested than WT mice, this is likely an artifact of earlier ejaculation in WT mice. We therefore conclude that sexual motivation was not different between WT and MC4RKO mice.

MC4R expressed only on Sim1 neurons rescues the sexual behavior changes seen in

MC4R null mice

Due to the position of the paraventricular nucleus of the hypothalamus downstream of POMC neurons, and its abundance of MC4 receptors, tbMC4Rsim1mice were generated to determine the location of the relevant MC4 receptors. Colocalization of

MC4R and Sim1 neurons was confirmed in the PVN (Fig. 3A), Nucleus of the Lateral

Olfactory Tract (NLOT) (Fig. 3B), which is a region of the amygdala (49), and slightly in the SON (Fig. 3C) using immunofluorescence, which was consistent with previous studies (6). In tbMC4Rsim1 mice, colocalization was confirmed in the PVN (Fig. 4A) and

NLOT (Fig. 4B) but not confirmed in the SON (Fig. 4C). tbMC4Rsim1 mice were paired with females at two months of age and sexual behavior was observed. Sim1-cre controls were not different from WT mice for mounting behavior (p=0.3468) or any other measure, so controls were represented by the WT group in all other figures. The tbMC4Rsim1 mice did not exhibit any altered behavior compared to wild type or sim1-cre controls, however, mounting attempts (p=0.002), intromission (p=0.0133), and latency to ejaculation (p=0.0049) were all significantly lower than MC4RKO mice (Fig. 5A-E, n=9-

11).

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MC4R expressed only on oxytocin neurons also rescues the sexual behavior changes seen in MC4R null mice

To further narrow down the neurocircuitry underlying this MC4R-mediated sexual behavior, a subset of Sim1 neurons, oxytocin neurons (25), were also observed for sexual behavior deficits. tbMC4Roxt mice were generated and immunofluorescence was used to confirm colocalization of MC4R and oxytocin neurons in Oxt-cre controls (Fig.

6A) and tbMC4Roxt mice (Fig. 6B). tbMC4Roxt mice were paired with females at two months of age and sexual behavior was observed. Because Oxt-cre mice were not different from WT mice in mounting (p=0.929), or any other measure, WT mice were used to represent controls for the remainder of the figures. tbMC4Roxt mice similarly did not exhibit any altered behavior compared to wild type or Oxt-cre controls, however, mounting attempts (p=0.0002), intromission (p=0.0025), and latency to ejaculation

(p=0.002) were significantly lower than MC4RKO mice (Fig. 7A-E, n=8-11).

The effect of MC4R on sexual function is independent of its role on metabolism

MC4 Receptors are known to be involved in the satiety pathway (2), so a metabolic profile was obtained from the tested mice the day following their sexual behavior testing, to determine whether the sexual behavior deficits could be attributed to altered metabolism. Nuclear magnetic resonance (NMR) revealed that MC4R manipulation did indeed affect weight gain. Fisher’s LSD multiple comparison test revealed a significant difference between WT and MC4RKO (p=0.0004) as well as between tbMC4Roxt and WT (p=0.0002), but not between WT and tbMC4Rsim1

(p=0.1103) (Fig. 8A). tbMC4Rsim1 weights were attenuated compared to both MC4RKO

(p=0.0309) and tbMC4Roxt (p=0.015) mice. This increase in weight was likely attributed

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to a similar increase in fat mass with Fisher’s LSD multiple comparison test revealing a strong difference between WT and MC4RKO mice (p<0.0001) as well as a smaller difference between WT and tbMC4Roxt (p=0.004) (Fig. 8B). Interestingly, the significant one-way ANOVA in lean mass at two months of age (F(3,37)=7.612, p=0.0004) was attributed primarily to an increase in tbMC4Roxt mice compared to WT (p<0.0001),

MC4RKO (p=0.0305), and tbMC4Rsim1 (p=0.0007), indicating that the weight gain in tbMC4Roxt mice is not just to fat gain, but also an increase in lean muscle (Fig. 8C).

MC4RKO mice also had a small significant increase in lean mass compared to WT

(p=0.0203). Fluid mass was similar to lean mass except there was no significant difference between MC4RKO and tbMC4Roxt mice (p=0.1444) (Fig. 8D). tbMC4Rsim1 mice showed an attenuated weight, fat content, lean mass, and fluid composition which is consistent with previous literature (39). A glucose tolerance test yielded no significance between groups (p=0.5508), suggesting that at two months of age, MC4RKO mice do not have a diabetic phenotype which would therefore not explain the sexual behavior deficits

(Fig. 8E-F). These results were somewhat surprising, as previous studies have reported hyperglycemia in MC4RKO mice as young as 10 weeks old (24, 41, 44). One possible explanation for this discrepancy is that these mice were on a C57Bl6 background whereas the mice in this study were on a mixed C57Bl6/Agouti background. C57Bl6 mice are known to be susceptible to the development of type II diabetes (43).

Due to the significant increase in weight and fat composition in MC4RKO, a series of correlations were computed to determine if there is a relationship between increased weight and altered sexual behaviors (Table 1). Number of mounts did show a significant correlation with weight, however, it is possible that this is due to the skewed

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nature of the normal sexual behavior of all normal weighted control animals. Therefore, correlations were also done only on mice with an obesity-prone genotype (MC4RKO, tbMC4Rsim1, and tbMC4Roxt) because while all of these mice are heavier, many of them could copulate normally. These correlations were no longer significant, suggesting that the MC4RKO is causing sexual deficits independent of weight gain.

Metabolic cages were utilized to examine how MC4R was influencing respiration and energy expenditure. At two months of age, MC4RKO mice did not have a clear change in vO2 (Fig. 9A-B), vCO2 (Fig. 9C-D), or energy expenditure (Fig. 9G-H) compared to controls, however, they did have a significantly lower respiratory exchange ratio (p=0.0431), indicating a stronger utilization of fat tissue for energy compared to controls (Fig. 9E-F). tbMC4Rsim1 mice were not different from MC4RKO or WT mice any measure except they had a lower RER compared to WT controls during the day

oxt (p=0.0013). tbMC4R mice had an increase in vO2, vCO2, and energy expenditure compared to all other groups.

The metabolic cages also provided information regarding activity of the mice. All genotypes with reduced MC4R expression were less active compared to controls (Fig.

10A-B), implying that MC4R involved in activity may be on neurons other than Sim1 and oxytocin neurons. This was consistent with previous studies that found a decrease in locomotor activity in MC4R null mice (13). Interestingly, despite the increased energy expenditure seen in the tbMC4Roxt mice, they had the lowest levels of activity. Sexual behavior deficits cannot be attributed to a lack of activity because these mice were able to perform sexually, despite having lower activity levels than MC4RKO mice. Food intake was increased in MC4RKO mice (p=0.0003) and tbMC4Roxt mice (p<0.0001) compared

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to WT and tbMC4Rsim1 mice (p=0.0473 and 0.0176 respectively) as expected (Fig. 10C).

Water intake was also increased in MC4RKO (p=0.0084) and tbMC4Roxt (p=0.0401) compared to WT mice (Fig. 10D). In summary, while MC4RKO mice ate more and were less active than WT mice, tbMC4Roxt were also less active and hyperphagic, but were able to perform normally in sexual behavior tests. We conclude that the sexual behavior changes in MC4RKO mice was independent of metabolic deficits.

Diet-induced obesity alone could not produce the sexual function deficits seen in

MC4RKO mice

To confirm that obesity by itself does not explain the MC4RKO sexual phenotype, a high fat diet control group (WT-HFD) was utilized. These mice were age and weight matched to MC4RKO mice. An NMR study confirmed that these mice had a metabolic phenotype consistent with MC4RKO mice (p=0.7917) (Fig. 11A). The two- month sexual behavior test of mice fed high fat diet showed mounting behavior

(p=0.0019), intromission (p=0.011), and latency to ejaculate (p=0.003) were reduced compared to MC4RKO mice and consistent with wild type controls (Fig. 11B, D, F).

Interestingly, there was a decrease in latency to mount in WT-HFD mice compared to

WT controls (p=0.0313) (Fig. 11C). There was no difference in the intromission:mount ratio across groups (Fig. 11E). Overall, weight gain alone was not responsible for the sexual deficits found in MC4RKO mice.

Sex hormones were not altered in MC4RKO mice

Serum was collected from mice at 3-4 months of age to assess hormone concentrations. Results showed that knocking out MC4R had no effect on serum LH,

FSH, the LH/FSH ratio, testosterone, estradiol, (Fig. 12A-E). Interestingly, there was a

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significant difference between the tbMC4Roxt serum LH and MC4RKO serum LH

(p=0.012), but the lack of a difference in the LH/FSH ratio or testosterone suggests that this may not have any physiological significance.

3.4 Discussion:

Previously, our lab and others have shown that 6-month-old MC4RKO male mice and mice with insensitive POMC neurons showed a delay in mounting behavior when paired with sexually receptive females (14, 48). This was interpreted as reduced interest in sexual behavior and this implied that MC4R must be involved in sexual interest. Our lab hypothesized that knocking out MC4R would show similar results in younger mice, however, we found a very different sexual phenotype. These mice actually had increased mounting behavior and no difference in latency to mount. Rather than attributing this behavior to an increase in sexual motivation, we noted the increased latency to ejaculate in MC4R null mice. Considering that mice lose interest in mounting after reaching ejaculation, mice that take longer to ejaculate, but have intact motivation, will continue to mount over a longer period of time. We thus interpret this data as an impairment in ejaculation, rather than an increase in motivation. This suggests that MC4Rs have a stronger role in regulating ejaculation, rather than interest, at two months of age. This is supported because previous studies of MC4RKO mice found an inability of these males to reach ejaculation (48).

Due to our initial hypothesis, that the involved MC4Rs were downstream of

POMC neurons, we tested mice in which we knocked in MC4R only on certain neurons, to see if that would recover the deficit. Using tbMC4Rsim1 and tbMC4Roxt mice, sexual

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behavior was restored to normal at two months of age. The paraventricular nucleus has previously been implicated in the central control of erectile function (28). The connection between oxytocin and melanocortins in regulating sexual behavior has been suggested by multiple sources (18, 19, 33) , but this is the most direct evidence to date that oxytocin neurons are involved in this melanocortin-mediated neurocircuitry.

Our results reveal a strong connection between MC4R on oxytocin neurons and ejaculation, although, somewhat surprisingly, not between these receptors and erectile function. Similar studies have found a decreased latency to ejaculate in rabbits and rats after administration of oxytocin (5, 15). Interestingly, another study using an oxytocin knock-out mouse model found that oxytocin was not necessary for sexual behavior (26).

This suggests that there may be alternative melanocortin-mediated pathways mediating ejaculatory function. Further studies will be needed to determine the involvement of other neurons. Our results support the notion that oxytocin may be an effective treatment for men who suffer from delayed ejaculation. Delayed ejaculation is not well-understood, although it is known to be distressing for the men who receive this diagnosis. The efficacy of pharmaceutical therapy for this disorder is still under investigation (1). One study administered oxytocin intranasally to men prior to masturbation, but latency to ejaculation was not significantly decreased (50). Another study found that intranasal oxytocin resulted in an increased latency to ejaculation compared to placebo (10). Both of these studies were done on healthy men, rather than men who suffer from delayed ejaculation. A third study administering intranasal oxytocin found that men reported more intense orgasm after treatment (8). Our results suggest that this treatment may warrant further exploration.

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These data also provide a novel interpretation of previous studies that examined

MC4R knock-out mice at 3-6 months of age (48). At 6 months of age, we found that

MC4R null mice indeed show an increased latency to mount, consistent with other findings. They also had impaired intromission efficiency and a reduced ability to ejaculate. One potential explanation for the discrepancies in motivation and erectile function is that MC4R are also involved in metabolism, so at an older age, obesity may become a much larger factor. When reducing the age-related obesity factor, it seems possible that central MC4R may be specifically impacting ejaculation. These data have been supported by findings that show that MC4R in the PVN are not involved in the erectogenic response (4). This is important to consider when developing therapeutic agents that target the melanocortin system. According to our results, treating a patient population that has erectile dysfunction with a melanocortin agonist may not be as effective as treating a population with ejaculation disorders. However, it is also important to consider that the melanocortin system may be tied to erectile dysfunction through pathways distinct from those that were studied here. Further research needs to be done to confirm these findings in animals as well as in humans.

To confirm that the sexual function of our mice was unaffected by the metabolic changes that accompany a lack of MC4R, we characterized the metabolic phenotype of each genotype. We hypothesized that if the metabolic phenotype in MC4RKO mice was uniquely impaired, then the sexual dysfunction seen in these mice may simply have been an indirect effect of altered metabolism. We were able to show that the metabolic effects of knocking out MC4R cannot explain the changes in sexual performance, as tbMC4Roxt also had an increase in obesity and fat mass, and actually had an increased energy

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expenditure profile despite having normal sexual function. This was a surprising finding, but may be due to the significant increase in lean mass, which all measures were normalized to. One previous study found that MC4RKO mice do not have a compensatory increase in activity or thermogenesis in response to high caloric intake

(12). It is possible that the increased energy expenditure in tbMC4Roxt mice indicates that

MC4R on oxytocin neurons is sufficient to restore one aspect of compensation for increased food intake. Another study in which oxytocin neurons were ablated revealed that mice fed high fat diet showed reduced energy expenditure, supporting a role for oxytocin in this metabolic function (52). The ablation of oxytocin neurons had no effect on food intake or activity, which is consistent with our findings. However, other studies have shown that oxytocin may have a role in exercise as oxytocin administration results in decreased tachycardia (9). Perhaps this effect was not seen in tbMC4Rsim1 mice because these mice did not have altered food intake. Conflicting results have been found regarding RER. Another study found that when RER was normalized to the lean body mass, as was done in the current studies, RER was increased in MC4R null mice, but when normalized to total weight, RER was decreased compared to controls (13). This contradicts our findings of reduced RER in all MC4R-altered genotypes. Overall, the metabolic cages revealed that MC4R is involved in RER, activity, and food intake.

Despite these alterations, tbMC4Roxt mice had a similar metabolic phenotype, with the exception of increased energy expenditure, but were able to perform normally in sexual behavior tests.

To determine how much of a factor the obesity alone played in sexual behavior, an age-matched, weight-matched HFD WT control was used, confirming that the delay in

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ejaculation in the MC4RKOs at a young age was due to the central control of the melanocortin 4 receptors rather than the increased adiposity. We propose that age may confound behavioral results and studies using such profound genetic manipulation may be better executed when mice are young. The weight of MC4RKO mice at two months of age is equivalent to the weight of WT mice at 6 months of age, and is not enough adiposity to have any significant effect on sexual function. However, MC4RKO mice can reach over 60g by six months of age, which could have a clear impact on mobility and sexual function. An alternative approach could be to use an inducible knock-out model, which would allow for mice to develop normally, and may help to clarify the specific roles of the melanocortin 4 receptor.

The melanocortin system plays a role in regulating blood pressure (21). Agonists of MC4R result in hypertension, although MC4R null mice have been found to have normal blood pressure despite their obesity (34). It has been hypothesized that this may be due to the regulation of sympathetic outflow. This is important to consider because erectile function is dependent on normal blood flow and autonomic activity. Because erectile function and ejaculation are known to be regulated by the autonomic system (28), it will be important to explore how this hypothalamic neurocircuitry is connected to sympathetic and parasympathetic pathways. There is evidence for neurons in the PVN, including oxytocin, to project to the brain stem and spinal cord autonomic systems (37,

38, 40).

In conclusion, we found that ejaculation in young male mice is mediated through melanocortin 4 receptors on Sim1, and more specifically, oxytocin neurons. Furthermore, we confirmed that this behavior was not confounded by the metabolic effects of MC4R.

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We highlight the importance of considering the effects of age and age-related obesity on studies investigating sexual behavior. This data may help to reconcile conflicting literature on sexual dysfunction as well as contributes to the existing knowledge of the neurocircuitry underlying ejaculation. Expanding upon this neurocircuitry may lead to better treatment options for men with sexual dysfunction.

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3.5 Figures:

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0 0 0 2 0 0 0 4 0 0 0 6 0 0 0 L a te n c y to E ja c u la tio n (s )

Figure 1. Sexual behavior in MC4RKO mice. Mounting attempts between wild-type and MC4RKO mice (n=11 WT and MC4RKO) was significantly different compared to WT mice (A). Latency to mount was not different between groups (B). Intromission number was also significantly different using an unpaired t test compared to WT mice (C). The ratio of successful intromission to mounting attempts which is an indicator of intromission efficiency was not significant (D). Latency to Ejaculation was significantly compared to WT mice (E). Across all groups, all but one MC4RKO mouse achieved ejaculation within the six hours filmed. There was a significant correlation between latency to ejaculate and mounting attempts (F).

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A A n o g e n ita l S n iff T im e B M o u n tin g A tte m p ts in 2 0 M in u te s

3 0 0 4 0 W T * M C 4 R K O 3 0

) 2 0 0

s

(

e

2 0

m i

T 1 0 0 1 0

0 0 C D M o u n tin g A tte m p ts fo r M ic e T h a t F a ile d % R e a c h e d E ja c u la tio n to R e a c h E ja c u la tio n in 2 0 M in u te s 1 .0 4 0 0 .8 3 0

t

n 0 .6

e c r 2 0

e 0 .4 P

1 0 0 .2

0 .0 0 Figure 2. Sexual motivation in MC4RKO mice is unimpaired compared to WT controls (N=11 per group). Time spent in the anogenital sniffing phase was not different between MC4RKO and WT mice (A). MC4RKO mice had increased mounting attempts within the first twenty minutes of sexual behavior compared to controls (B). There is a trend for fewer MC4RKO mice reaching ejaculation within the first twenty minutes of behavior compared to WT mice (C). When examining mounting attempts only in mice that did not reach ejaculation within the first twenty minutes, there is no longer a significant difference (D).

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Sim1-cre (Td-Tomato) MC4R Overlay A

PVN

B

NLOT

C

SON

Figure 3. Immunofluorescence to show colocalization of Sim1-cre with MC4R in Sim1- cre mice. Sim1-cre neurons are identified using a Td-tomato reporter (red), MC4R is visible using a green fluorescent labeled secondary antibody, and the overlay of the two is visible in yellow. The top panels (A) show the PVN. The NLOT and SON, which are also Sim1 regions, both show some colocalization with MC4R (B-C). Each image was taken at 40X magnification. A scale for size is located in the bottom right corner of each image.

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Sim1-cre (Td-Tomato) MC4R Overlay

A

PVN

B

NLOT

C

SON

Figure 4. Immunofluorescence to show colocalization of Sim1-cre with MC4R in tbMC4Rsim1 mice. Sim1-cre neurons are identified using a Td-tomato reporter (red), MC4R is visible using a green fluorescent labeled secondary antibody, and the overlay of the two is visible in yellow. The top panels (A) show the PVN, the NLOT is shown in the second set of panels (B), and the SON, which did not show colocalization with MC4R was shown in the last set of panels (C). All images were taken at a 40X magnification.

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M o u n tin g A tte m p ts A B L a te n c y to M o u n t 1 5 0 * * * W T 4 0 0 * * * * S im 1 c re M C 4 R K O 3 0 0 1 0 0

s im 1 )

tb M C 4 R s

(

e 2 0 0

m

5 0 i T 1 0 0 0 0

In tro m is s io n N u m b e r In tro m is s io n : M o u n t R a tio C D 5 0 * * * 1 .0

4 0 0 .8

3 0 0 .6

o i

t a

2 0 R 0 .4

1 0 0 .2

0 0 .0

L a te n c y to E ja c u la tio n E 4 0 0 0 * * * * *

3 0 0 0

)

s (

e 2 0 0 0

m

i T

1 0 0 0

0

Figure 5. tbMC4Rsim1 mice had normal sexual behavior compared to controls. Mounting Attempts between wild-type, Sim1-cre mice, tbMC4Rsim1, and MC4RKO mice (n=9-11) was significantly different due to increased mounting attempts in MC4RKO compared to all other groups (A). Due to no difference between WT and Sim1-cre, future results are reported with WT group for ease of visualizing results. Latency to mount was not different between groups (B). Intromission number was also significantly reduced in tbMC4Rsim1 mice compared to MC4RKO mice (C). The intromission to mount ratio (intromission efficiency) showed no significant difference across groups (D). Latency to Ejaculation (from first mount) was significantly decreased in tbMC4Rsim1 mice compared with MC4RKO mice (E).

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Oxytocin-cre (GFP) MC4R Overlay

A

B

Figure 6. Colocalization of oxytocin neurons with MC4R. Colocalization was seen between oxytocin neurons (green) and MC4R (red) in both Oxt-cre-GFP mice (20X magnification) (A) and tbMC4Roxt mice (40X magnification) (B).

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A M o u n tin g A tte m p ts B L a te n c y to F irs t M o u n t

1 5 0 4 0 0 * * * * W T O x t-c re * * * * * * 3 0 0 M C 4 R K O

1 0 0 )

o x t s (

tb M C 4 R

e 2 0 0

m i

5 0 T 1 0 0

0 0

C In tro m is s io n N u m b e r D In tro m is s io n :M o u n t R a tio

5 0 0 .8 * * * * 4 0 0 .6

3 0

o i

t

0 .4 a

2 0 R

0 .2 1 0

0 0 .0

E L a te n c y to E ja c u la tio n

4 0 0 0 * * * * *

3 0 0 0

)

s

(

e 2 0 0 0

m i

T 1 0 0 0

0 Figure 7. Sexual behavior of tbMC4Roxt was comparable to WT mice. Mounting attempts between wild-type, Oxt-cre mice, tbMC4Roxt, and MC4RKO mice (n=8-11) was significantly different one-way ANOVA, with a post-hoc test revealing that all groups had significantly reduced mounting attempts compared to MC4RKO mice (A). Due to no difference between WT and Oxt-cre in any parameter, future results are reported with WT group for ease of visualizing results. Latency to mount was not different between any groups (B). Intromission number was also significantly increased in MC4RKO compared to all other groups (C). The intromission:mount ratio, or number of times intromission was reached during mounting attempts, was not significantly different across groups (D). Latency to Ejaculation (from first mount) was significantly different by one-way ANOVA, with Fisher’s LSD multiple comparison tests revealing that tbMC4Roxt mice were comparable to WT mice (E).

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A W e ig h t B F a t M a s s # 4 0 # 8 # * * * * * * W T * * * * M C 4 R K O

3 0 ) 6

) g

s im 1 ( * * g

( tb M C 4 R

s t

o x t s h

2 0 tb M C 4 R a 4

g

i

M

e

t

a W 1 0 F 2

0 0

C L e a n M a s s D F lu id M a s s # # # 8 2 5 ^ # # * * * * * * * 2 0 *

) 6 * *

g

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s g

1 5 (

s

a d

i 4

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u

l

n 1 0

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a e

L 2 5

0 0

GTT E F A re a U n d e r th e C u rv e 500 4 0 0 0 0 WT 400 MC4RKO 3 0 0 0 0

300 C

U 2 0 0 0 0 200 A

1 0 0 0 0

Glucose (mg/dL) Glucose 100

0 0 0 50 100 150 Time (min) Figure 8. MC4R has an effect on weight gain. Nuclear Magnetic Resonance (NMR) showed a significant difference between groups at 2 months of age using a one-way ANOVA (N=8-11). MC4RKO and tbMC4Roxt mice had significantly increased weight gain compared to WT (*) and tbMC4Rsim1 (#) mice (A). Fat mass was similarly increased in MC4RKO and tbMC4Roxt mice compared to WT with a smaller difference between MC4RKO and tbMC4Rsim1 (B). Interestingly, the significant one-way ANOVA in lean mass at two months of age was attributed primarily to tbMC4Roxt with tbMC4Roxt significantly increased compared to all groups including MC4RKO (^) (C). Fluid mass was similarly increased in tbMC4Roxt compared to WT and tbMC4Rsim1 mice as well as MC4RKO and WT mice (D). Glucose Tolerance Testing (GTT) showed no significant difference between WT and MC4RKO at 2-3 months of age. Multiple t-tests were used to analyze differences across time-points and an unpaired t-test was used to analyze differences in area under the curve (N=10). *=compared to WT, ^=compared to MC4RKO, #=compared to tbMC4Rsim1.

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Table 1. Correlations between weight and sexual behavior measures.

X Variable Y Variable R square P value

Two Month Weight (All Mice) Mounting Attempts 0.1171 0.006**

Two Month Weight (Overweight Mice) Mounting Attempts 0.06755 0.1508

Two Month Weight (All Mice) Latency to Mount 0.01417 0.3528

Two Month Weight (Overweight Mice) Latency to Mount 0.01375 0.5227

Two Month Weight (All Mice) Intromission 0.02477 0.218

Two Month Weight (Overweight Mice) Intromission 0.03371 0.3144

Two Month Weight (All Mice) Latency to Ejaculation 0.05485 0.0789

Two Month Weight (Overweight Mice) Latency to Ejaculation 0.01439 0.5278

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s im 1 A W T tb M C 4 R 7 0 0 0 B o x t C W T 8 0 0 0 M C 4 R K O tb M C 4 R # # M C 4 R K O ^ ^

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[ 4 0 0 0

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2

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3 0 0 0 0 0 1 0 2 0 3 0 D a y N ig h t H o u r

C D # 6 0 0 0 6 0 0 0 ^ *

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g *

k

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6 0 0 0 1 0 2 0 3 0 D a y N ig h t H o u r Figure 9. Metabolic cages normalized to lean body mass revealed an interesting effect of a significantly increased vO2 (A-B), vCO2 (C-D), and “heat” or energy expenditure in the tbMC4Roxt during the day and night (G-H) compared to all other groups using a one-way ANOVA test between time and genotype (n=8-9). Panels in the left column represent the measures as they changed over the course of the day, while panels in the right column represent averages during the day and night. Significant interactions and main effects were found for all measures. The RER, a measure of vO2 over vCO2 showed that all MC4R altered groups were significantly lower than WT (E-F) but when averaged, this was not significant. *=compared to WT, ^=compared to MC4RKO, #=compared to tbMC4Rsim1.

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A B 2 0 0 0 2 5 0 0 W T M C 4 R K O 1 5 0 0 2 0 0 0 s im 1

tb M C 4 R

y t

1 0 0 0 y i

o x t t 1 5 0 0 i

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v * *

t

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C D 6 * * * * *#* * 6 # W T ) * *

g *

)

(

g

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(

y y s im 1 a

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/ D

/ o x t e

n tb M C 4 R

k

e

a

t

t

a

n

I

E

2 2

r

d

e o

t

o

a

F W 0 0

Figure 10. Metabolic cages also measured activity, food intake, and water intake of mice. Activity is represented across time (A) and as an average during the day and during the night (B). tbMC4Roxt showed the least activity at night, although MC4RKO also showed decreased activity compared to WT controls. A one-way ANOVA showed increased food intake attributed to tbMC4Roxt and MC4RKO but not tbMC4Rsim1 (C). Water intake was also increased in MC4RKO and tbMC4Roxt mice (D). *=compared to WT, #=compared to tbMC4Rsim1. N=8-9 for all tests.

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A W e ig h t (g ) B M o u n tin g A tte m p ts

4 0 1 5 0 W T * * * * * M C 4 R K O 3 0 * * * * * W T -H F D 1 0 0

2 0

5 0 1 0

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C L a te n c y to M o u n t D In tro m is s io n N u m b e r 4 0 0 5 0 * * *

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0 0

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0 .6 )

s

o

(

i

t e

a 2 0 0 0

R m

0 .4 i

T

1 0 0 0 0 .2

0 .0 0

Figure 11. Sexual behavior between wild-type (n=11), WT mice on high fat diet (WT- HFD, n=5), and MC4RKO mice (n=11). WT-HFD mice were a comparable weight to MC4RKO mice when tested (A). WT-HFD mounting attempts were significantly reduced compared to MC4RKO (B). Latency to mount was not significantly different across groups, but there was a significant reduction in latency to first mount in WT-HFD mice compared to WT controls (C). Intromission number was also significantly increased in MC4RKO mice compared to WT-HFD mice (D). There was no difference in the intromission:mount ratio across groups (E). Latency to Ejaculation (from first mount) was significantly increased in MC4RKO compared to WT-HFD mice, but comparable between WT-HFD and WT mice (F).

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A S e ru m L H B S e ru m F S H 0 .5 *

W T 1 0 0

)

) L 0 .4 M C 4 R K O L

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m

m /

/ s im 1 g

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6 0

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L

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m

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E S e ru m E s tra d io l

6

)

L m

/ 4

g

p (

l

o

i d

a 2 r

t

s E

0

Figure 12. Serum LH (A), FSH (B), the LH/FSH ratio (C), testosterone (D), and estradiol (E) concentrations were not different using a one-way ANOVA (N=8-9). There was, however, a difference between tbMC4Roxt serum LH and MC4RKO serum LH using a Fisher’s LSD multiple comparison test.

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Chapter 4

Hypothalamic Melanocortin 4 Receptors Affect Lordosis in Female Mice Independent of Metabolic Changes

*Manuscript intended for publication

4.1 Introduction:

Although sexual dysfunction has been extensively studied in men, the literature is much more scarce for women. Available studies have found that approximately 40% of women in the United States reported any sexual problem (27, 28, 30, 37, 48) and 22% experienced distress as a result of their sexual problem (30). The true prevalence and incidence of female sexual dysfunction is difficult to determine in part due to a lack of standardized definitions and assessment tools for the female sexual response cycle (6, 7,

20, 21, 23, 33, 34, 52).

There is a distinct lack of effective options for treating sexual dysfunction in women. Androgens, phosphodiesterase 5 inhibitors, and other treatments approved for men have been explored with little success in women (35, 39, 44, 60). Recently, a drug has been approved by the FDA for women with hypoactive sexual desire disorder

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(HSDD) (14). This drug, flibanserin, is a 5HT1A agonist/5HT2A antagonist (9) but its lack of specificity in the central nervous system may lead to unwanted side effects (18,

24, 39).

Another central target that has received attention in treating female sexual dysfunction is the melanocortin pathway (60). Bremelanotide, a melanocortin 3,4 receptor agonist, has been investigated for improving sexual desire in women (11, 13,

45). MC4R, found primarily in the brain (49, 54), has shown promise for being involved in the central regulation of sexual function (12, 43). Bremelanotide increased both arousal and sexual satisfaction in women (11). Unfortunately, intranasal bremelanotide has adverse side-effects in women, such as increasing blood pressure, so clinical trials were discontinued (31, 45, 57). One way to bypass these side-effects is by developing more specific therapeutic agents. To this end, it is imperative to explore which downstream pathways are controlled by melanocortins.

Melanocortin 4 Receptor is known to be found in the paraventricular nucleus (49,

54). The paraventricular nucleus of the hypothalamus has been implicated in female sexual behavior (19, 32, 43, 58). Oxytocin neurons, found exclusively in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the hypothalamus have also been implicated in female sexual behavior (3, 16, 46).

Previous findings in our lab have implicated MC4R expressed on Sim1 neurons, which are found primarily in the PVN and amygdala, as well as MC4R expressed on oxytocin neurons in mediating sexual behavior in male mice. Due to the known involvement of these neurons in female sexual behavior, we hypothesized that there is a

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melanocortin-mediated neurocircuitry involving Sim1 neurons and, more specifically, oxytocin neurons regulating sexual receptivity in female mice.

In these studies, we explore the role of MC4R in regulating sexual activity in female mice. We show that MC4R null mice show reduced sexual receptivity to males.

When MC4R is expressed only on Sim1 neurons or oxytocin neurons, lordosis behavior resembles that of wild-type controls. We confirm that these findings are independent of the effect of MC4R on metabolism. These results elucidate a role and pathway for the melanocortin system in female sexual behavior, which may inform future investigations into therapeutic options in the treatment of female sexual dysfunction.

4.2 Methods:

Animal Production and Care:

All mice used in this study were kept in accordance with the University’s IACUC guidelines. MC4R knock-out mice were purchased from The Jackson Laboratory (loxTB

Mc4r, 006414). These mice contain a floxed transcription blocker in the promoter for the

MC4R gene. The transcription blocker prevents MC4R expression, but tissue-specific cre expression results in the removal of the transcription blocker, and thus the expression of

MC4R. To target the paraventricular nucleus, we utilize Sim1-cre mice. Sim1 is a transcription factor that is necessary for the development of the PVN (36, 55), and studies have used this is a way to target this nucleus (5, 47, 61). In our previously established mouse model, mice expressing cre on Sim1 neurons, and homozygous for the floxed

MC4R gene would express MC4R only on Sim1-expressing neurons (tbMC4Rsim1).

Oxytocin neurons that expressed cre were used to generate mice that expressed MC4R only on those neurons (tbMC4Roxt). Mice were bred such that cre-expression was

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heterozygous, which has been shown to be sufficient in the past and would limit the chance for an unintended cre-specific phenotype. Genotyping was confirmed by sending a small piece of tail to Transnetyx, Inc. for automated genotyping using qPCR.

Mice were housed in DLAR facilities where they were given ad libidum water on a 12:12 light dark cycle. Food was given ad libidum.

Sexual Behavior Studies:

Female mice were ovariectomized around 5-6 weeks of age and given a week to recover. Beginning at week 7-8, these female mice were paired with experienced male mice four separate times in order to gain sexual experience. This was done to ensure that lordosis would be measurable, as previous studies in mice have found that inexperienced females are much less likely to exhibit lordosis (4, 26). Females were hormonally primed using

100µL of subcutaneous β-estradiol-3-benzoate in sesame oil (200µg/mL) 48 hours prior to sexual behavior, followed by 125 µL of intraperitoneal progesterone (4mg/mL) 7 hours prior to pairing to prime females (17). Pairing was done with experienced male mice from

8pm-9am, during their normal period of activity.

The fifth pairing was filmed (DVR Swann 4500 and T850 Day and Night Security

Camera security system) and analyzed for sexual behaviors during 8pm-2am. The primary measure of female sexual receptivity in rodents is lordosis. Criteria for lordosis was all four paws of the female firmly planted on the ground with the front half of the mouse pushed up off the ground. The reported measure of lordosis is the lordosis quotient, which is the percentage of time lordosis is displayed when mounted by a male. Lordosis behavior shown in the absence of a male mounting the female was not counted. Videos were blinded and one observer scored all videos to reduce inter-observer variability.

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Metabolic Testing:

MC4R null mice have a well-established phenotype in the literature as obese mice

(5). The early age of sexual behavior was determined because our lab has discovered that, due to the metabolic profile of MC4R null mice, after reaching adulthood, the obesity becomes a confounding factor in testing sexual function. Glucose tolerance test (GTT), nuclear magnetic resonance (NMR) for fat content, and metabolic cages were measured to assess the metabolic profile of these mice.

The morning after sexual behavior testing, Nuclear Magnetic Resonance (NMR) was used to assess fat content (BrukerOptics). At 9-12 weeks old, a glucose tolerance test

(GTT) was done. The morning of the GTT, mice were fasted for 6 hours on alpha-dri bedding. Following the fast, baseline glucose levels were obtained by measuring tail-vein blood with AlphaTRAK 2 (ADW Diabetes) test strips and glucometer. A 2g/kg dosage of dextrose was given intraperitoneally and subsequent blood glucose levels were measured at 15, 30, 45, 60, 90, and 120 minutes post-injection.

Metabolic cages (Columbus Instruments’ Comprehensive Lab Animal Monitoring

System) were used to determine energy expenditure, food intake, and activity. Mice were give a day to acclimate to the metabolic cages before measurements were taken. All measurements were taken and averaged over a three-day period.

Diet Induced Obesity:

For DIO tests, wild-type littermates were given High Fat Diet (HFD)

(OpenSource Diets, 60% fat content) instead of standard chow at week 5. Mice were weighed weekly until their weights were comparable to MC4RKO mice at the time of their sexual behavior testing. DIO mice went through all tests as previously described.

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Serum Hormone Concentrations:

Submandibular blood samples were taken at week 10-13. Blood was spun down in the centrifuge for 10 minutes at 4°C at 4472 rcf. Serum was collected and stored in -80°C until analysis. LH/FSH was measured by UVA using a multiplex assay.

Immunohistochemistry:

Upon euthanasia, mice were perfused with 4% Paraformaldehyde and the brain was obtained. Brains were transitioned to a 10% sucrose, 20% sucrose, and finally a 30% sucrose solution in PBS. The brain was sectioned in 35-40µm slices and then stored in cryoprotectant (20% glycerol and 30% ethylene glycol in PBS) until immunohistochemistry was performed in order to identify the location of MC4R in the hypothalamus and its co-localization with Sim1 and oxytocin. To verify that there are

MC4R on Sim1 neurons in the PVN, brain sections were labeled overnight with rabbit anti-MC4R (Abcam ab24233) and then exposed to a Donkey anti-rabbit IgG Alexa Fluor

488 secondary antibody (Invitrogen, A21206). Td-tomato was used as a cre-reporter and the fluorescence was visible via confocal without the aid of an additional antibody. To verify that there are MC4R on oxytocin neurons in the PVN, brain sections were labeled overnight with rabbit anti-MC4R (Abcam ab24233) and chicken anti-GFP (Aves Labs

Inc, IgY0511FP12), because the oxytocin-cre expression was paired with GFP. Sections were exposed to Alexa Fluor 488 and 594–conjugated secondary antisera (donkey anti rabbit Alexa Fluor 594, A21207, Life Technologies; goat anti chicken alexa fluor 388,

A11039, life technologies) and observed using a confocal microscope (Leica).

Statistical Analysis:

All data in figures are represented as mean ± SEM. Parametric, two-tailed

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unpaired t-tests were used to compare two groups, and a one-way ANOVA was used to compare more than two groups followed by Fisher’s LSD multiple comparison post-tests.

Correlation tests were used to determine the relationship between two continuous variables with results reported as R2 values. Statistical significance was defined as p<0.05. Data in bar graphs presented as mean ±SEM (95% confidence interval). In figure legends, *, p<0.05; **, p<0.01; ***p<0.001.

4.3 Results:

MC4R null females exhibit reduced sexual receptivity compared to WT controls

Ovariectomized, experienced females were primed with estradiol and progesterone and paired with experienced, control males at two months of age. Pairing was done at 8pm at night and mice were filmed for six hours. The video was analyzed for a lordosis quotient, as an indication of sexual receptivity. MC4RKO females had a decreased lordosis quotient compared to wild-type controls (Fig. 1; N=8-9, p=0.0012). This is an indication that

MC4RKO mice are less sexually receptive than controls, which implicates the MC4R in mediating sexual receptivity.

MC4R expression exclusively on Sim1 neurons resulted in a lordosis quotient comparable to controls

After establishing a phenotype of impaired lordosis in female MC4RKO mice, we hypothesized that the MC4R may be on Sim1 neurons, because our lab had found a similar phenotype in male mice. To test this hypothesis, we tested tbMC4Rsim1 mice that have Sim1 neuron-specific expression of MC4R. Mice with MC4R present only on Sim1 neurons had a sexual behavior phenotype consistent with that of controls and had a significantly higher lordosis quotient compared to MC4RKO mice (Fig. 1, p=0.0009). These results suggest

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that MC4 receptors that are on Sim1 neurons play a role in mediating sexual receptivity.

Although these transgenic mice have been well-established in the literature, we confirmed colocalization of MC4R and sim1 neurons using immunofluorescence in Sim1-cre mice

(Fig. 2A-B) and tbMC4Rsim1 mice (Fig. 2C-D).

MC4R expression exclusively on Oxytocin neurons resulted in a lordosis quotient comparable to controls

Due to the large population of Sim1 neurons represented by oxytocin neurons (25), we further tested mice with an oxytocin neuron-specific expression of MC4R to determine the role of these neurons in this melanocortin-mediated behavior. tbMC4Rsim1 mice have been confirmed in previous studies, but there is conflicting literature regarding the presence of MC4R on oxytocin neurons. We confirmed the colocalization of oxytocin neurons and

MC4R using immunofluorescence in both Oxt-cre mice (Fig. 3A) and tbMC4Rtb mice (Fig.

3B). Mice with MC4R present only on oxytocin neurons (tbMC4Roxt) also had a sexual behavior phenotype consistent with that of controls and had a significantly higher lordosis quotient compared to MC4RKO mice (p=0.0077) (Fig. 4). These results are consistent with findings that paraventricular oxytocin neurons are activated during sexual intercourse (16).

MC4R mediates sexual behavior independent of metabolism deficits

To determine the effect of altered metabolism, we acquired an extensive metabolic profile on these mice. Consistent with previous studies, MC4RKO mice did have statistically increased weight gain compared to controls (p=0.0005) (5), as well as a significant increase in fat mass (p=0.0038), lean mass (p=0.0016), and fluid (p=0.0022)

(Fig. 5A-D). Both tbMC4Rsim1 as well as tbMC4Roxt mice also had significantly increased weight, fat mass, lean mass, and fluid, despite previous research finding an attenuate weight

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gain phenotype in tbMC4Rsim1 mice (5). Previously, it has been reported that age-matched mice that did not undergo ovariectomy showed MC4R null mice had greater weight compared to tbMC4Rsim1 females. It is possible that since these studies were done approximately a month post-OVX surgery, that these mice were affected by post-op weight loss. There was no correlation between weight and lordosis quotient (Fig. 5E). MC4RKO mice had normal glucose tolerance (Fig. 6A-B; p=0.4478) which was consistent with previous studies that found no hyperglycemia in female MC4R null mice (53). Other female MC4R null mice have been found to have hyperglycemia but found that when mice were pair-fed to attenuate the weight gain, hyperglycemia was reduced, suggesting that hyperglycemia is secondary to the increased adiposity (51). A reduced metabolic rate was found in MC4R null female mice, suggesting that hyperphagia may be secondary to increased adiposity (51). These results suggest that the sexual behavior phenotype is independent of metabolic changes.

Metabolic cages were used to test the respiratory exchange rate, energy expenditure, activity, food and water intake. The tbMC4Roxt mice had the biggest change

oxt in respiration compared to WT mice. tbMC4R mice had an increase in vO2 (Fig. 7A-B) compared to both WT and MC4RKO mice during the day and night. tbMC4Roxt mice also had an increase in vCO2 (Fig. 7C-D) compared to WT across the 24 hours, and compared to tbMC4Rsim1 mice during the day but not at night. However respiratory exchange rate

(vO2/vCO2) (Fig. 7E-F) was not different between any groups. Energy expenditure, or

“heat,” was significantly increased in tbMC4Roxt compared to WT mice during both day and night, and increased compared to MC4RKO mice during the day (Fig. 7G-H). Total activity was found to be significantly decreased in all genotypes compared to WT during

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the day, but tbMC4Rsim1 mice had a significant attenuation compared to MC4RKO mice.

At night, when mice are most active, only MC4RKO and tbMC4Roxt mice had significantly reduced activity compared to WT mice and this was significantly attenuated in tbMC4Rsim1 mice (Fig. 8A-B). Food intake, as expected, was significantly increased in all genotypes compared to WT mice (Fig. 8C). Interestingly tbMC4Roxt mice also ate more than tbMC4Rsim1 mice. tbMC4Roxt also drank more water compared to all groups but tbMC4Rsim1 drank less than MC4RKO mice (Fig. 8D). Overall, while MC4RKO mice had some altered metabolism parameters, tbMC4Roxt was more strongly affected. Because tbMC4Roxt were able to perform normally in the sexual behavior tests despite their altered metabolism, it was concluded that these MC4R-mediated effects did not result in the impaired sexual performance in MC4RKO mice at two months of age.

Mice fed high fat diet had normal sexual function at two months of age

To compare the effect of weight on the MC4R null mice, WT mice were fed a high fat chow diet after weaning until their weights were comparable to MC4R null mice (Fig.

9A; p=0.6941). These mice went through the same sexual behavior paradigms and were found to exhibit a lordosis quotient comparable to WT mice and significantly higher than

MC4RKO mice (Fig. 9B; p=0.0007). These data suggest that the sexual behavioral changes can not be attributed to weight gain alone.

Serum LH/FSH was comparable in all experimental mice

Although these mice were ovariectomized, and therefore, their sex hormones were controlled exogenously, their serum LH and FSH concentrations were measured, as well as the LH/FSH ratio, and found not to be different between groups (Fig. 10A-C).

4.4 Discussion:

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In our studies, we found that MC4R null mice had a reduced lordosis quotient compared to controls. This is contrary to findings by Pfaus et al., who found no effect of an MC4R agonist, Bremelanotide, on lordosis, but did find an increase in solicitation behaviors in rats (43). Because all animals tested had a 100% lordosis quotient, however, it is possible that their hormonal priming regimen was too high to allow for any meaningful difference to be detected. It is also unclear how well bremelanotide crossed the blood brain barrier through subcutaneous administration in these studies. Another study showed that a melanocortin receptor agonist did increase lordosis behavior in rats when injected centrally, although this article suspected the involvement of MC3R in the

VMH rather than MC4R in the PVN (12). It is possible that these two receptor populations are involved in the same neurocircuitry, but further research will need to be done to elucidate the connection between the two.

Using genetic manipulation to selectively express MC4R in Sim1 and oxytocin neurons, we found that oxytocin neurons that express MC4R are important for regulating lordosis in female mice. Support for Sim1 neurons such as paraventricular nucleus neurons being involved in the female sexual response has been seen using viral tracing studies (19, 32, 58). In fact, Gelez et al. found immunoactivity in both MC4R and oxytocin neurons after a tracer virus was injected into the clitoris and vagina of female rats (19). Oxytocin has been previously implicated in regulating lordosis (22, 40, 41), but a direct connection between the melanocortin and oxytocin pathway had not been previously elucidated. While our studies implicate this pathway in sufficiency, oxytocin receptor knock-out studies suggest that this pathway is likely not necessary for lordosis

(29).

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MC4R is known to be involved in the sensation of satiety after eating (1).

Because MC4RKO mice are known to cause obesity, there was a possibility that the metabolic effects of MC4R may have had an influence on the changes in sexual behavior.

To test this, mice were assessed for a number of metabolic parameters including body mass composition, glucose tolerance, respiratory exchange rate, energy expenditure, activity, and food intake. Even at two-months of age, food intake was markedly decreased in MC4RKO and tbMC4Roxt mice. Despite this increase in food intake, there was no metabolic compensation in MC4RKO mice in terms of activity or energy expenditure. Surprisingly, although tbMC4Roxt mice had low levels of activity, energy expenditure was increased. This suggests a potential role for MC4R on oxytocin neurons in energy expenditure. Studies have confirmed a role for oxytocin neurons in energy expenditure, but only to compensate for increased caloric intake (59). tbMC4Rsim1 exhibited an expected intermediate phenotype in terms of food intake, activity, and energy expenditure. This attenuation was expected based on previous findings (5, 61).

Because our female mice were ovariectomized, a full hormonal profile was not assessed. However, the LH/FSH examination was found to be normal at 2 months of age.

Previous studies found that LH concentrations as well as ovarian histology was altered between 4 and 7 months of age in MC4R null mice (10). As fertility is known to decline in mice at this age, it is likely that these mice would have an altered profile at a later time-point.

The literature on central regulation of female sexual behavior is complex, but one key hypothalamic region that appears to be involved in lordosis is the ventral medial nucleus of the hypothalamus (VMH) (2, 12, 15, 16, 32). Interestingly, there is evidence

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that supports a circuit between oxytocin and the VMH (32, 56). It is well-documented that the estrogen-gating of sexual receptivity seems to be located in the VMH (58). Considering the normal receptivity in the control mice in our studies when hormonally primed, but altered activity in MC4RKO mice despite receiving estradiol and progesterone, it is possible that the role of the VMH in lordosis may be downstream of the PVN. Other brain regions known to receive oxytocin projections from the PVN include the spinal cord, the

MPOA, the amygdala, the nucleus tractus solitarius (NTS) and the dorsal motor nucleus of the vagus nerve (DMV) (50). There is also evidence for MC3R and MC4R involvement in the lordosis quotient in the medial preoptic nucleus (19, 38).

Many parallels have been drawn between female lordosis and male ejaculatory control (58). This is an interesting dichotomy to consider because our lab has found that

MC4RKO mice also have a longer latency to ejaculation. Although humans do not exhibit the same lordosis postural reflex that rodents do, it has been suggested that the closest analogous experience in humans is orgasm (42). In support of this concept, one study administering intranasal oxytocin reported that participants experienced increased contentment after sexual intercourse (8). While medications targeting the melanocortin system are tested in the treatment of disorders of sexual desire and arousal, our studies suggest that there may be a role for these drugs in improving orgasm or overall satisfaction.

In conclusion, we found that lordosis in female mice is due at least in part to MC4 receptors on Sim1 neurons, representing the PVN and the amygdala. Furthermore, we confirm a subpopulation of Sim1 neurons, oxytocin neurons, that are involved in the regulation of lordosis behavior. Obtaining a metabolic profile of these mice suggested that the impact of MC4R on sexual behavior is independent of its impact on metabolism. These

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findings support the investigation of drugs that target the melanocortin and oxytocin systems in the treatment of sexual disorders. Future elucidation of this neurocircuitry will have strong implications for narrowing down more specific targets for female sexual dysfunction.

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4.5 Figures:

L o rd o s is Q u o tie n t

0 .8 * * W T * * * * * * S im 1 -c re 0 .6 M C 4 R K O

s im 1 td M C 4 R 0 .4

0 .2

0 .0

Figure 1. Lordosis quotient between wild-type, Sim1-cre mice, MC4RKO, and tbMC4Rsim1 mice (n=8-9) was significantly different with one-way ANOVA. Post-hoc tests revealed that all groups had a significantly higher lordosis quotient compared to MC4RKO, suggesting that MC4R only on Sim1 neurons is sufficient for normal sexual receptivity.

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Sim1-cre (Td-Tomato) MC4R Overlay

A

B

C

D

Figure 2. Immunofluorescence to show colocalization of Sim1-cre with MC4R in Sim1- cre and tbMC4Rsim1 mice. Sim1-cre neurons are identified using a Td-tomato reporter (red), MC4R is visible using a green fluorescent labeled secondary antibody, and the overlay of the two is visible in yellow. The top panels (A) show the PVN in Sim1-cre mice. The next panel shows the amygdala (B). Below that, images were taken from tbMC4Rsim1 mice. The PVN and NLOT, which is a region of the amygdala, both show faint colocalization with MC4R (C-D). Scale is located on the bottom right corner of each image.

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Oxytocin-cre (GFP) MC4R Overlay

A

3V 3V 3V

B

3V 3V 3V Figure 3. Oxytocin and MC4R are co-localized. 3V= Third Ventricle. Green represents oxytocin-GFP neurons. Red represents MC4R. Yellow represents colocalization of the two. Top two rows of panels are of the paraventricular nucleus in oxt-cre-GFP brains (A) and tbMC4Roxt brains (B). Scale is located in the bottom right corner of each image.

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L o rd o s is Q u o tie n t 0 .8 * * * * * W T O x t-c re 0 .6 * * M C 4 R K O

o x t td M C 4 R 0 .4

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A Glucose Tolerance Test B A re a U n d e r th e C u rv e 500 3 0 0 0 0 WT 400 MC4RKO 2 0 0 0 0

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Figure 9. Lordosis quotient between wild-type (n=9), WT mice on high fat diet (n=5), and MC4RKO mice (n=9) was significantly different with a one-way ANOVA. Fisher’s LSD multiple comparison test was used to determine that lordosis quotient was significantly higher in WT-HFD compared to MC4RKO mice but not different from WT mice.

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Chapter 5

Summary of Findings and Future Directions

Sexual dysfunction is highly prevalent in the populations of both men and women

(15), but due to stigma and a lack of understanding of the pathophysiology underlying sexual dysfunction, there are few adequate treatment options available. Melanocortins have been investigated in the treatment of sexual dysfunction (19), but new insights into the neurocircuitry mediating melanocortin involvement would assist in developing therapeutic agents. The goal of this research was to identify central melanocortin pathways mediating sexual behavior. These studies carefully elucidated how these pathways influence specific types of sexual behavior, which is important for determining the efficacy of drugs in clinical trials. Confounding factors such as age and obesity were also investigated. Finally, we determined that these pathways affect both male and female mice, increasing the possibility that these therapeutic targets should be investigated in both men and women.

In the first study, we confirmed that six-month-old male mice lacking MC4R have sexual dysfunction in the form of erectile dysfunction and an inability to ejaculate (23).

Using tbMC4RSim1 mice, we proved that MC4R on Sim1 neurons is sufficient for erectile function and an ability to ejaculate comparable to control mice. We found that central

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αMSH administration had the unexpected effect in tbMC4RSim1 mice of causing them to lose the ability to ejaculate. We speculate that because αMSH has an affinity for receptors other than MC4R, there may be some compensatory mechanism in these mice causing the inhibition of ejaculation. Identifying this mechanism may provide further understanding of the interplay between inhibiting and stimulating ejaculation, as both premature ejaculation and delayed ejaculation are disorders that men suffer from. Overall, the elucidation that MC4R on Sim1 neurons play a significant role in sexual function allows us to focus on specific neuronal targets for central therapeutics. We are particularly interested in the paraventricular nucleus of the hypothalamus, as it contains numerous

MC4 receptors (20), is one of the major regions of Sim1 neurons (16, 22), and is a known projection site of POMC neurons which release melanocortins (25). Furthermore, lesioning studies and tracing studies specifically implicate the involvement of the PVN in sexual behavior (4, 9, 13). This neuroanatomical information is imperative for further study of the pathways underlying sexual behavior and the subsequent targets that can be used for developing treatments.

Due to the significant obesity in the MC4RKO mice at six months of age, we repeated the study in two-month-old mice, when obesity is much less of a factor. We found that these mice had a much clearer sexual phenotype of delayed ejaculation but normal erectile function. This supports and clarifies the findings from the first study, as well as stresses the importance of considering age when studying sexual function in both animals and humans. The finding that erectile function was not affected by young

MC4RKO mice is supported in the literature by studies that found that MC4R did not seem to be the receptor responsible for melanocortin-induced erections (24). Not only did

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we confirm that MC4 receptors on Sim1 neurons recover ejaculation latencies, but we further found that MC4 receptors on oxytocin neurons also recover this deficit. Sim1 is a marker for a variety of different types of neurons, but a haploinsufficiency of Sim1 results in a dramatic reduction in oxytocin neurons (12). The finding that MC4R on oxytocin neurons recovers the same deficits that were recovered in tbMC4Rsim1 mice suggests that oxytocin could be the major player represented by our Sim1 findings. In addition to elucidating this neurocircuitry, we confirmed that MC4R results in a metabolic phenotype of obesity due to weight gain and reduced activity and increased food intake. Through correlational studies and comparing between genotypes, we concluded that this metabolic phenotype was not confounding the sexual behavior at two months of age. tbMC4Rsim1 mice had an attenuated metabolic phenotype, as expected.

However, tbMC4Roxt mice displayed a metabolic phenotype that was, if anything, more severe than MC4R null mice in terms of eating behavior and activity. Interestingly, tbMC4Roxt mice had increased energy expenditure, perhaps as compensation for lack of activity and excessive calorie intake. This is supported by previous findings that have shown that oxytocin does have a role in diet-induced energy expenditure, but not food intake or activity (26). This finding may warrant future studies into the role of oxytocin in energy expenditure.

Finally, we explored these melanocortin pathways in two-month-old female sexual behavior. We found that MC4RKO mice had a reduced lordosis quotient, indicating less sexual receptivity. Expressing MC4R exclusively on Sim1 or oxytocin neurons restored lordosis quotients to percentages consistent with control mice. We confirmed that these findings were not confounded by the MC4R-mediated metabolism

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phenotype. This finding is important in implicating melanocortins and oxytocin as therapeutic targets for female sexual function. Previous studies investigating the role of melanocortins in female sexual behavior have found evidence for its involvement in regulating sexual desire and arousal (10, 11, 18). Our findings suggest that melanocortins and downstream targets such as oxytocin may improve female orgasm and sexual satisfaction. This is not the first time that oxytocin has been implicated in the female orgasm (5, 14). This finding is particularly interesting in light of our male findings which emphasize the role of melanocortins and oxytocin in ejaculation, rather than erection.

Other studies have found evidence for oxytocin’s involvement in male ejaculation (1, 2).

Further understanding what lies downstream of melanocortins in regulating sexual dysfunction will be key in the development of therapies without unwanted side-effects.

Melanocortins are clearly involved in metabolic regulation (3, 8, 21), regulating the cardiovascular system (17), and there is even evidence for a melanocortin role in depression and anxiety (6, 7). Clarifying how these various pathways intersect may allow for the development of treatments that are more specific for sexual dysfunction.

Alternatively, this knowledge may allow for the treatment of both sexual dysfunction and some of the common co-morbidities associated with sexual dysfunction.

Collectively, we have provided both confirmatory and new insights into the role of MC4R in sexual behavior. We clarify that MC4R appears to be primarily involved in ejaculatory function in males, although in older mice, erectile function may also be affected. We also found new evidence for the involvement of MC4R in sexual receptivity. Using genetic manipulation, we selectively expressed MC4R on specific tissues. Using this tool, we determined that MC4R on both Sim1 neurons, and more

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specifically, oxytocin neurons, play an important role in regulating ejaculation in males and lordosis in females. These findings implicate melanocortins and oxytocin in sexual function which may allow for more targeted development of therapies. These findings may also assist in the development of clinical trials of such therapeutic agents, because the parameters under investigation are important assessing the efficacy of a drug, as our studies highlight the importance of ejaculation latencies and female sexual satisfaction, whereas many drugs are being marketed as treating sexual desire and arousal.

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Chapter 2

1. Abdel-Hamid IA, Elsaied MA, and Mostafa T. The drug treatment of delayed ejaculation. Translational andrology and urology 5: 576-591, 2016.

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Chapter 3

1. Abdel-Hamid IA, Elsaied MA, and Mostafa T. The drug treatment of delayed ejaculation. Translational andrology and urology 5: 576-591, 2016.

2. Adan RA, Tiesjema B, Hillebrand JJ, la Fleur SE, Kas MJ, and de Krom M. The MC4 receptor and control of appetite. British journal of pharmacology 149: 815-827, 2006.

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3. Argiolas A, Melis MR, and Gessa GL. Yawning and penile erection: central dopamine-oxytocin-adrenocorticotropin connection. Annals of the New York Academy of Sciences 525: 330-337, 1988.

4. Argiolas A, Melis MR, Murgia S, and Schioth HB. ACTH- and alpha-MSH- induced grooming, stretching, yawning and penile erection in male rats: site of action in the brain and role of melanocortin receptors. Brain research bulletin 51: 425-431, 2000.

5. Arletti R, Bazzani C, Castelli M, and Bertolini A. Oxytocin improves male copulatory performance in rats. Hormones and behavior 19: 14-20, 1985.

6. Balthasar N, Dalgaard LT, Lee CE, Yu J, Funahashi H, Williams T, Ferreira M, Tang V, McGovern RA, Kenny CD, Christiansen LM, Edelstein E, Choi B, Boss O, Aschkenasi C, Zhang CY, Mountjoy K, Kishi T, Elmquist JK, and Lowell BB. Divergence of melanocortin pathways in the control of food intake and energy expenditure. Cell 123: 493-505, 2005.

7. Barberis C, Mouillac B, and Durroux T. Structural bases of vasopressin/oxytocin receptor function. The Journal of endocrinology 156: 223-229, 1998.

8. Behnia B, Heinrichs M, Bergmann W, Jung S, Germann J, Schedlowski M, Hartmann U, and Kruger TH. Differential effects of intranasal oxytocin on sexual experiences and partner interactions in couples. Hormones and behavior 65: 308-318, 2014.

9. Braga DC, Mori E, Higa KT, Morris M, and Michelini LC. Central oxytocin modulates exercise-induced tachycardia. American journal of physiology Regulatory, integrative and comparative physiology 278: R1474-1482, 2000.

10. Burri A, Heinrichs M, Schedlowski M, and Kruger TH. The acute effects of intranasal oxytocin administration on endocrine and sexual function in males. Psychoneuroendocrinology 33: 591-600, 2008.

11. Butler AA, and Cone RD. The melanocortin receptors: lessons from knockout models. Neuropeptides 36: 77-84, 2002.

12. Butler AA, Marks DL, Fan W, Kuhn CM, Bartolome M, and Cone RD. Melanocortin-4 receptor is required for acute homeostatic responses to increased dietary fat. Nature neuroscience 4: 605-611, 2001.

13. Chen AS, Metzger JM, Trumbauer ME, Guan XM, Yu H, Frazier EG, Marsh DJ, Forrest MJ, Gopal-Truter S, Fisher J, Camacho RE, Strack AM, Mellin TN, MacIntyre DE, Chen HY, and Van der Ploeg LH. Role of the melanocortin-4 receptor in metabolic rate and food intake in mice. Transgenic research 9: 145-154, 2000.

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14. Faulkner LD, Dowling AR, Stuart RC, Nillni EA, and Hill JW. Reduced melanocortin production causes sexual dysfunction in male mice with POMC neuronal insulin and leptin insensitivity. Endocrinology 156: 1372-1385, 2015.

15. Fjellstrom D, Kihlstrom JE, and Melin P. The effect of synthetic oxytocin upon seminal characteristics and sexual behaviour in male rabbits. Journal of reproduction and fertility 17: 207-209, 1968.

16. Frye CA, and Vongher JM. Progesterone has rapid and membrane effects in the facilitation of female mouse sexual behavior. Brain research 815: 259-269, 1999.

17. Gades NM, Jacobson DJ, McGree ME, St Sauver JL, Lieber MM, Nehra A, Girman CJ, and Jacobsen SJ. Longitudinal evaluation of sexual function in a male cohort: the Olmsted county study of urinary symptoms and health status among men. The journal of sexual medicine 6: 2455-2466, 2009.

18. Gelez H, Poirier S, Facchinetti P, Allers KA, Wayman C, Alexandre L, and Giuliano F. Neuroanatomical evidence for a role of central melanocortin-4 receptors and oxytocin in the efferent control of the rodent clitoris and vagina. The journal of sexual medicine 7: 2056-2067, 2010.

19. Giuliano F. Control of penile erection by the melanocortinergic system: experimental evidences and therapeutic perspectives. Journal of andrology 25: 683-691, 2004.

20. Giuliano F, and Allard J. Dopamine and male sexual function. European urology 40: 601-608, 2001.

21. Greenfield JR, Miller JW, Keogh JM, Henning E, Satterwhite JH, Cameron GS, Astruc B, Mayer JP, Brage S, See TC, Lomas DJ, O'Rahilly S, and Farooqi IS. Modulation of blood pressure by central melanocortinergic pathways. The New England journal of medicine 360: 44-52, 2009.

22. Hatzimouratidis K. Epidemiology of male sexual dysfunction. American journal of men's health 1: 103-125, 2007.

23. Hatzimouratidis K, Amar E, Eardley I, Giuliano F, Hatzichristou D, Montorsi F, Vardi Y, and Wespes E. Guidelines on male sexual dysfunction: erectile dysfunction and premature ejaculation. European urology 57: 804-814, 2010.

24. Huszar D, Lynch CA, Fairchild-Huntress V, Dunmore JH, Fang Q, Berkemeier LR, Gu W, Kesterson RA, Boston BA, Cone RD, Smith FJ, Campfield LA, Burn P, and Lee F. Targeted disruption of the melanocortin-4 receptor results in obesity in mice. Cell 88: 131-141, 1997.

25. Kublaoui BM, Gemelli T, Tolson KP, Wang Y, and Zinn AR. Oxytocin deficiency mediates hyperphagic obesity of Sim1 haploinsufficient mice. Molecular endocrinology (Baltimore, Md) 22: 1723-1734, 2008. 115

26. Lazzari VM, Becker RO, de Azevedo MS, Morris M, Rigatto K, Almeida S, Lucion AB, and Giovenardi M. Oxytocin modulates social interaction but is not essential for sexual behavior in male mice. Behavioural brain research 244: 130-136, 2013.

27. Lewis RW, Fugl-Meyer KS, Corona G, Hayes RD, Laumann EO, Moreira ED, Jr., Rellini AH, and Segraves T. Definitions/epidemiology/risk factors for sexual dysfunction. The journal of sexual medicine 7: 1598-1607, 2010.

28. Marson L, Platt KB, and McKenna KE. Central nervous system innervation of the penis as revealed by the transneuronal transport of pseudorabies virus. Neuroscience 55: 263-280, 1993.

29. McCabe MP, Sharlip ID, Lewis R, Atalla E, Balon R, Fisher AD, Laumann E, Lee SW, and Segraves RT. Incidence and Prevalence of Sexual Dysfunction in Women and Men: A Consensus Statement from the Fourth International Consultation on Sexual Medicine 2015. The journal of sexual medicine 13: 144-152, 2016.

30. Melis MR, Melis T, Cocco C, Succu S, Sanna F, Pillolla G, Boi A, Ferri GL, and Argiolas A. Oxytocin injected into the ventral tegmental area induces penile erection and increases extracellular dopamine in the nucleus accumbens and paraventricular nucleus of the hypothalamus of male rats. The European journal of neuroscience 26: 1026-1035, 2007.

31. Michaud JL, Rosenquist T, May NR, and Fan CM. Development of neuroendocrine lineages requires the bHLH-PAS transcription factor SIM1. Genes & development 12: 3264-3275, 1998.

32. Mitchell KR, Mercer CH, Ploubidis GB, Jones KG, Datta J, Field N, Copas AJ, Tanton C, Erens B, Sonnenberg P, Clifton S, Macdowall W, Phelps A, Johnson AM, and Wellings K. Sexual function in Britain: findings from the third National Survey of Sexual Attitudes and Lifestyles (Natsal-3). Lancet (London, England) 382: 1817-1829, 2013.

33. Modi ME, Inoue K, Barrett CE, Kittelberger KA, Smith DG, Landgraf R, and Young LJ. Melanocortin Receptor Agonists Facilitate Oxytocin-Dependent Partner Preference Formation in the Prairie Vole. Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology 40: 1856-1865, 2015.

34. Ni XP, Butler AA, Cone RD, and Humphreys MH. Central receptors mediating the cardiovascular actions of melanocyte stimulating hormones. Journal of hypertension 24: 2239-2246, 2006.

35. Nicolosi A, Moreira ED, Jr., Shirai M, Bin Mohd Tambi MI, and Glasser DB. Epidemiology of erectile dysfunction in four countries: cross-national study of the prevalence and correlates of erectile dysfunction. Urology 61: 201-206, 2003.

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36. Safarinejad MR, and Hosseini SY. Salvage of sildenafil failures with bremelanotide: a randomized, double-blind, placebo controlled study. The Journal of urology 179: 1066-1071, 2008.

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42. Succu S, Sanna F, Cocco C, Melis T, Boi A, Ferri GL, Argiolas A, and Melis MR. Oxytocin induces penile erection when injected into the ventral tegmental area of male rats: role of nitric oxide and cyclic GMP. The European journal of neuroscience 28: 813-821, 2008.

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46. Thorve VS, Kshirsagar AD, Vyawahare NS, Joshi VS, Ingale KG, and Mohite RJ. Diabetes-induced erectile dysfunction: epidemiology, pathophysiology and management. Journal of diabetes and its complications 25: 129-136, 2011.

47. Tolson KP, Gemelli T, Gautron L, Elmquist JK, Zinn AR, and Kublaoui BM. Postnatal Sim1 deficiency causes hyperphagic obesity and reduced Mc4r and 117

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48. Van der Ploeg LH, Martin WJ, Howard AD, Nargund RP, Austin CP, Guan X, Drisko J, Cashen D, Sebhat I, Patchett AA, Figueroa DJ, DiLella AG, Connolly BM, Weinberg DH, Tan CP, Palyha OC, Pong SS, MacNeil T, Rosenblum C, Vongs A, Tang R, Yu H, Sailer AW, Fong TM, Huang C, Tota MR, Chang RS, Stearns R, Tamvakopoulos C, Christ G, Drazen DL, Spar BD, Nelson RJ, and MacIntyre DE. A role for the melanocortin 4 receptor in sexual function. Proceedings of the National Academy of Sciences of the United States of America 99: 11381-11386, 2002.

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Chapter 4

1. Adan RA, Tiesjema B, Hillebrand JJ, la Fleur SE, Kas MJ, and de Krom M. The MC4 receptor and control of appetite. British journal of pharmacology 149: 815-827, 2006.

2. Aou S, Oomura Y, and Yoshimatsu H. Neuron activity of the ventromedial hypothalamus and the medial preoptic area of the female monkey during sexual behavior. Brain research 455: 65-71, 1988.

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5. Balthasar N, Dalgaard LT, Lee CE, Yu J, Funahashi H, Williams T, Ferreira M, Tang V, McGovern RA, Kenny CD, Christiansen LM, Edelstein E, Choi B, Boss O, Aschkenasi C, Zhang CY, Mountjoy K, Kishi T, Elmquist JK, and Lowell BB. Divergence of melanocortin pathways in the control of food intake and energy expenditure. Cell 123: 493-505, 2005.

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7. Basson R, Berman J, Burnett A, Derogatis L, Ferguson D, Fourcroy J, Goldstein I, Graziottin A, Heiman J, Laan E, Leiblum S, Padma-Nathan H, Rosen R, Segraves K, Segraves RT, Shabsigh R, Sipski M, Wagner G, and Whipple B. Report of the international consensus development conference on female sexual dysfunction: definitions and classifications. The Journal of urology 163: 888-893, 2000.

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13. Diamond LE, Earle DC, Heiman JR, Rosen RC, Perelman MA, and Harning R. An effect on the subjective sexual response in premenopausal women with sexual arousal disorder by bremelanotide (PT-141), a melanocortin receptor agonist. The journal of sexual medicine 3: 628-638, 2006.

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17. Frye CA, and Vongher JM. Progesterone has rapid and membrane effects in the facilitation of female mouse sexual behavior. Brain research 815: 259-269, 1999.

18. Gao Z, Yang D, Yu L, and Cui Y. Efficacy and Safety of Flibanserin in Women with Hypoactive Sexual Desire Disorder: A Systematic Review and Meta-Analysis. The journal of sexual medicine 12: 2095-2104, 2015.

19. Gelez H, Poirier S, Facchinetti P, Allers KA, Wayman C, Alexandre L, and Giuliano F. Neuroanatomical evidence for a role of central melanocortin-4 receptors and oxytocin in the efferent control of the rodent clitoris and vagina. The journal of sexual medicine 7: 2056-2067, 2010.

20. Georgiadis JR, and Kringelbach ML. The human sexual response cycle: brain imaging evidence linking sex to other pleasures. Progress in neurobiology 98: 49-81, 2012.

21. Giraldi A, Rellini A, Pfaus JG, Bitzer J, Laan E, Jannini EA, and Fugl- Meyer AR. Questionnaires for assessment of female sexual dysfunction: a review and proposal for a standardized screener. The journal of sexual medicine 8: 2681-2706, 2011.

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16. Michaud JL, Rosenquist T, May NR, and Fan CM. Development of neuroendocrine lineages requires the bHLH-PAS transcription factor SIM1. Genes & development 12: 3264-3275, 1998.

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19. Shadiack AM, Sharma SD, Earle DC, Spana C, and Hallam TJ. Melanocortins in the treatment of male and female sexual dysfunction. Current topics in medicinal chemistry 7: 1137-1144, 2007.

20. Siljee JE, Unmehopa UA, Kalsbeek A, Swaab DF, Fliers E, and Alkemade A. Melanocortin 4 receptor distribution in the human hypothalamus. European journal of endocrinology 168: 361-369, 2013.

21. Ste Marie L, Miura GI, Marsh DJ, Yagaloff K, and Palmiter RD. A metabolic defect promotes obesity in mice lacking melanocortin-4 receptors. Proceedings of the National Academy of Sciences of the United States of America 97: 12339-12344, 2000. 125

22. Tolson KP, Gemelli T, Gautron L, Elmquist JK, Zinn AR, and Kublaoui BM. Postnatal Sim1 deficiency causes hyperphagic obesity and reduced Mc4r and oxytocin expression. The Journal of neuroscience : the official journal of the Society for Neuroscience 30: 3803-3812, 2010.

23. Van der Ploeg LH, Martin WJ, Howard AD, Nargund RP, Austin CP, Guan X, Drisko J, Cashen D, Sebhat I, Patchett AA, Figueroa DJ, DiLella AG, Connolly BM, Weinberg DH, Tan CP, Palyha OC, Pong SS, MacNeil T, Rosenblum C, Vongs A, Tang R, Yu H, Sailer AW, Fong TM, Huang C, Tota MR, Chang RS, Stearns R, Tamvakopoulos C, Christ G, Drazen DL, Spar BD, Nelson RJ, and MacIntyre DE. A role for the melanocortin 4 receptor in sexual function. Proceedings of the National Academy of Sciences of the United States of America 99: 11381-11386, 2002.

24. Vergoni AV, Bertolini A, Mutulis F, Wikberg JE, and Schioth HB. Differential influence of a selective melanocortin MC4 receptor antagonist (HS014) on melanocortin-induced behavioral effects in rats. European journal of pharmacology 362: 95-101, 1998.

25. Wang D, He X, Zhao Z, Feng Q, Lin R, Sun Y, Ding T, Xu F, Luo M, and Zhan C. Whole-brain mapping of the direct inputs and axonal projections of POMC and AgRP neurons. Frontiers in neuroanatomy 9: 40, 2015.

26. Wu Z, Xu Y, Zhu Y, Sutton AK, Zhao R, Lowell BB, Olson DP, and Tong Q. An obligate role of oxytocin neurons in diet induced energy expenditure. PloS one 7: e45167, 2012.

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