Regulation of Kisspeptin-expressing neurons and stimulatory mode of action of Kisspeptin in Immortalized Hypothalamic Cell Models

Transcription

1 Regulation of Kisspeptin-expressing neurons and stimulatory mode of action of Kisspeptin in Immortalized Hypothalamic Cell Models Zoey Friedman A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Physiology University of Toronto Copyright by Zoey Friedman 2013

2 Regulation of Kisspeptin-expressing neurons and stimulatory mode of action of Kisspeptin in Immortalized Hypothalamic Cell Models Zoey Friedman Abstract Fertility is a highly regulated process dependent on the orchestration of hypothalamic neuropeptides and peripheral hormones, which converge on GnRH neurons in the HPG axis. Kisspeptin and its receptor, Gpr54 have emerged as fundamental gatekeepers of reproduction, acting upstream of GnRH neurons. Kiss neurons have been found to express estrogen receptors, Gpr54 and the GnIH receptor, Gpr147. We have generated immortalized, murine hypothalamic cell lines to investigate mechanisms of gene transcription in kisspeptin-expressing neuronal cell models. We show using qrt-pcr and western blot analyses that kisspeptin-expressing neuronal models are targets for gonadal steroids and hypothalamic neuropeptides. Further, using a novel GnRH-secreting cell line, we report that GnRH neurons expressing Gpr54 and Gpr47 are stimulated by kisspeptin, although suppressed by a kisspeptin/gnih cotreatnent. Overall, these studies expand our knowledge of the regulation of Kiss neurons by estradiol, kisspeptin and GnIH, improving our understanding of kisspeptin as a modulator of GnRH neurons. ii

3 Acknowledgments Most importantly, I would like to thank Dr. Denise Belsham, my supervisor. Thank-you for providing me with the opportunity to work in your lab; it has been both a privilege and a wonderful learning experience. While I learned numerous scientific skills and methods, how to write scientifically and how to work with other scientists, I also was able to learn about myself, and develop as a person. Throughout the time I spent in the lab, I improved my organizational skills, time-management skills and communication skills all of which will benefit me for the rest of my life and in any career field. Dr. Belsham, you are an excellent role model for your students, teaching good work ethic and proper scientific conduct. You are involved in so many different things, whether it be the University of Toronto, The University Health Network, the Endocrine Society, or one of your other affiliations it is clear you are very passionate about what you do, and you are driven to succeed. Your work ethic is inspiring to all of us, and you are a great a motivator. I would like to thank you for developing and maintaining such a welcoming and comfortable, although demanding and stimulating research environment in your lab. While I encountered many obstacles while pursuing my degree, you have taught me to persevere, and I have been able to achieve success. I would like to also thank my Supervisory committee members, Dr. Theodore Brown and Dr. Mark Palmert. Throughout the past two years, you have provided me with novel insight, guidance, and you're your honest opinions. You have truly been fundamental in my journey. Of equal importance, I owe a huge thank-you to all of my colleagues in the Belsham lab! You have all been amazing to work with, and I cannot imagine getting through this without you! From giving me advice and guidance, to many fun times we have shared together, you are the cause for my positive attitude and the root of the Belsham Lab Spirit. I would like to thank all of you for making my Masters degree not only a wonderful learning experience, but also for making it an enjoyable time in my life. Last but not least, I would like to thank all of my family and friends for supporting me and believing in me throughout this long, demanding and sometimes stressful journey. Had it not been for your support, understanding, and love, I would not be where I am today! iii

4 TABLE OF CONTENTS List of tables and figures viii List of abbreviations.x Chapter 1: General Introduction General Introduction The Hypothalamic-Pituitary-Gonadal (HPG) Axis Gonadotropin-releasing Hormone (GnRH) Neurons Regulators of HPG axis and GnRH neurons Estrogens Estrogen Receptors Other Steroid Hormones Glucocorticoid Hormones Neurotransmitters RFamide peptides (GnIH and Kisspeptin) Kisspeptin Discovery of Kisspeptin and the Kiss-1R Gpr Kisspeptin and Gpr54 expression Kisspeptin neurons in the ARC and AVPV nuclei ARC KNDy neurons AVPV Kisspeptin neurons Signaling pathways activated by kisspeptin Kisspeptin activity in the hypothalamus Kisspeptin activity in the pituitary Kisspeptin and HPG axis in reproduction Kisspeptin activation at puberty Kisspeptin and the menstrual cycle 27 iv

5 1.6 Regulation of kisspeptin by afferent signals Gonadal steroid hormones Metabolic factors Circadian and seasonal changes GnIH Immortalized hypothalamic cell models for the study of neuroendocrine function Immortalized adult mouse hypothalamic cell lines (mhypoa-xx) Immortalized adult non clonal GnRH cell model (mhypoa-gnrh/gfp) Study Hypothesis and Aims...35 Chapter 2: Materials and Methods Cell culture and reagents Reverse transcriptase-polymerase chain reaction (RT-PCR) Quantitative real-time PCR Western Blot Analysis Statistical Analysis.43 Chapter 3: Sex Steroid Regulation of Estrogen Receptors and Gpr54 Gene Expression in Female Immortalized Hypothalamic Kisspeptin-expressing Cell Lines Abstract Introduction Results mhypoa-50 and mhypoa-55 neurons express Kiss1 and Gpr54, as well as ERs and other reproductive peptides and receptors E2 regulates Gpr54 and ERs gene expression in mhypoa-50 and mhypoa-55 neuronal models In the mhypoa-50 cell line, induction of mrna expression is abolished with ERα v

6 and ERβ antagonists, while the induction is lost with ERβ and Gpr30 antagonists in the mhypoa-55 neurons Discussion. 57 Chapter 4: Hypothalamic Neuropeptide Kisspeptin and GnIH Regulate Gene Expression and MAPK Signaling Pathways in Immortalized Hypothalamic Kisspeptin-expressing Cell Lines Abstract Introduction Results mhypoa-50 and mhypoa-55 neurons express Kiss1, Gpr54, and Gpr147 as well as other reproductive peptides and receptors Kiss-10 peptide treatment regulates Kiss1 and Gpr54 mrna expression in mhypoa-50 and mhypoa-55 cell lines Kiss-10 treatment activates MAPK intracellular signaling cascades in mhypoa-50 and mhypoa-55 cell lines GnIH peptide treatment regulates Kiss-1 and Gpr54 mrna expression in mhypoa- 50 and mhypoa-55 cell lines Discussion..74 Chapter 5: Kisspeptin and GnIH-mediated regulation of GnRH mrna levels in a Novel GnRH-secreting Cell Model Abstract Introduction Results..84 vi

7 5.3.1 Characterization of Gpr54 and Gpr147 mrna expression in mhypoa-gnrh/gfp neurons Kiss-10 and GnIH c-fos expression in mhypoa-gnrh/gfp neurons Kiss-10 mediates induction of GnRH mrna expression in mhypoa-gnrh/gfp cell line Kiss-10 and GnIH co-treatment mediates a suppression of GnRH mrna expression in mhypoa-gnrh/gfp cell line Discussion..88 Chapter 6: General Discussion Overall Conclusions Study Limitations Future Directions Concluding Remarks..109 References vii

8 List of Tables and Figures Figure 1.1. A schematic of the mammalian hypothalamic-pituitary-gonadal (HPG) axis.4 Figure 1.2. Schematic illustration of the proteolytic processing of Kiss-1 gene to generate active kisspeptin peptides.15 Figure 1.3 Schematic summarizing the current mechanism through which kisspeptin neurons are regulated by estrogen in the HPG axis Figure 1.4. Schematic illustration of the signal transduction pathways activated following kisspeptin/gpr54 binding 24 Figure 1.5 Schematic summarizing the mechanisms through which Kisspeptin and GnIH may participate in the regulation of the HPG axis..31 Table 2.1. List of primers used for one-step PCR and quantitative RT-PCR Figure 3.1: Characterization of the gene expression profile of kisspeptin-expressing, mhypoa-50 and mhypoa-55 neurons 50 Figure 3.2: Effect of Estradiol treatment on ER-α and Gpr54 transcript levels in the mhypoa-50 neurons.. 52 Figure 3.3. Effect of Estradiol treatment on ER-α, ER-β and Gpr54 transcript levels in the mhypoa-55 neurons 54 Figure 3.4. Effects of estrogen receptor antagonists on estradiol-mediated induction of ER-α and Gpr54 mrna expression in mhypoa-50 neurons 56 Figure 3.5. Effects of estrogen receptor antagonists on estradiol-mediated induction of ER-α, ER-β and Gpr54 mrna expression in mhypoa-55 neurons at 4 h and 24 h..58 Figure 4.1. Effect of Kiss-10 treatment on Kiss-1 and Gpr54 transcript levels in the mhypoa-50 neurons 69 Figure 4.2. Effect of Kiss-10 treatment on Kiss-1 and Gpr54 transcript levels in the mhypoa-55 neurons 70 viii

9 Figure 4.3. Kisspeptin phosphorylates ERK1/2 protein but not Akt protein in mhypoa-50 and mhypoa-55 cell lines.72 Figure 4.4. Kisspeptin modifies phosphorylation status of p38 protein in mhypoa-50 and mhypoa-55 cell lines.73 Figure 4.5. Effect of GnIH treatment on Kiss-1 and Gpr54 transcript levels in the mhypoa-50 neurons..75 Figure 4.6. Effect of GnIH treatment on Kiss-1 and Gpr54 transcript levels in the mhypoa-55 neurons..77 Figure 5.1. Gene expression profile of an adult-derived, non-clonal GnRH-secreting cell line, mhypoa-gnrh/gfp 85 Figure 5.2. Kisspeptin induces c-fos mrna expression in the mhypoa-gnrh/gfp neurons..87 Figure 5.3. Kisspeptin-mediated regulation of GnRH mrna expression in mhypoa- GnRH/GFP neuronal cells 89 Figure 5.4. Kisspeptin/GnIH cotreatment-mediated regulation of GnRH mrna expression in mhypoa- GnRH/GFP neuronal cells..91 ix

10 List of Abbreviations AC Act D ACTH ANOVA AR ARC AVPV bp camp CHO CNS CRE DMH DNA dntp DRB E2 EDTA ERα ERβ ERK FBS FSH GABA GCs adenylyl cyclase actinomycin D adrenocorticotropic hormone analysis of variance androgren receptor arcuate nucleus anteroventral periventricular base pair cyclic adenosine monophosphate Chinese hamster ovary central nervous system camp response element dorsomedial hypothalamus deoxyribonucleic acid deoxyribonucleotide triphosphate 5,6-Dichlorobenzimidazole riboside estradiol ethylenediaminetatraacetic acid estrogen receptor alpha estrogen receptor beta extracellular signal-regulated kinase fetal bovine serum follicle-stimulating hormone γ -aminobutyric acid glucocorticoids x

11 GnIH GnRH GnRH-GFP GPCR GR GT1 HH HPA HPG ICV IV kda Kiss-1 KNDy LH MAPK ME mirna mrna mpoa MS MSDS NKB NTC OVLT OVX gonadotropin-inhibitory hormone gonadotropin-releasing hormone GnRH-green fluorescence protein G-protein coupled receptor glucocorticoid receptor GnRH T-antigen hypogonadotropic hypogonadism hypothalamic-pituitary-adrenal hypothalamic-pituitary-gonadal intracerebroventricular intravenous kilodaltons Kisspeptin gene Kisspeptin/Neurokinin-B/Dynorphin neurons luteinizing hormone mitogen-activated protein kinase median eminence microrna messenger RNA medial preoptic area medial septum medial septum/diagonal band of Broca neurokin-b non-template control organum vasculosum of lamina terminalis ovarioectomized xi

12 P PR PBS PCR progesterone progesterone receptor phosphate buffer solution polymerase chain reaction PKC protein kinase C PLC phospholipase C PVN RFRP RNA RT-PCR SCN paraventricular nucleus Argenine (R)-phenylalanine (F) amide-related peptide ribonucleic acid real-time polymerase chain reaction suprachiasmatic nucleus SV40 simian virus 40 T-Ag TF T Antigen transcription factor xii

13 CHAPTER 1: GENERAL INTRODUCTION 1

14 1.1 General introduction The GnRH neuronal system functions as the central integrator and ultimate effector of the hypothalamic-pituitary-gonadal (HPG) axis [1]. Adequate pulsatile GnRH secretion is required for both attainment and maintenance of reproductive functions throughout reproductive life [2-4]. At the level of the hypothalamus, a complex network of neuroendocrine circuitries and pathways are essential for the correct timing and functioning of the HPG axis, ultimately leading to reproductive success. However, due to the interconnectivity and redundancy of particular neuronal pathways, pathophysiologies can arise from perturbations at any level of these central nervous system (CNS) circuitries, presenting as a concern in reproductive maturation and success [5-13]. Despite our improved knowledge of the GnRH neuronal system, the stimulatory neuronal systems upstream of GnRH neurons have remained rather ambiguous, until the recent discovery of kisspeptin and its receptor, Gpr54 [14, 15]. Today, kisspeptin and Gpr54 are universally recognized as critical players in the control of key aspects of reproductive development and function, ranging from neonatal sexual differentiation, to regulation of GnRH and gonadotropin secretion, to the metabolic gating of puberty and adult fertility [16, 17]. While numerous in vivo studies have explored the importance of the Kiss1-Gpr54 system, there exists a paucity of in vitro studies, namely due to the complexity and heterogeneity of the hypothalamus, making investigation of the molecular mechanisms governing kisspeptin and its signal transduction pathways difficult. In order to address these concerns, the central aim of this thesis is to elucidate the mechanisms of action of afferent regulators (including 17β-estradiol, kisspeptin and GnIH) of hypothalamic kisspeptin-expressing neurons and in turn, determine the effect of kisspeptin on hypothalamic GnRH neurons downstream. To complete these studies, we have utilized novel, immortalized rodent hypothalamic cell lines generated in our lab. 1.2 The Hypothalamic-Pituitary-Gonadal (HPG) Axis 2

15 The hypothalamic-pituitary gonadal (HPG) axis, like all other endocrine systems, is dependent on an array of interconnected networks of communicating neuronal and organ systems. In 1932, it was first suggested that a scheme of reciprocal interplay between the pituitary and the testes existed [18]. In the years that followed, more work in the field lead to the understanding that the CNS was also largely involved in reproductive mechanisms, particularly specialized neuronal circuits in the hypothalamus [18-23]. It became apparent that a hierarchical system exists between the hypothalamus, the pituitary gland, and the gonads, and thus the HPG axis was established. Located at the pinnacle position of the rodent HPG axis is the anterior hypothalamus, where a small population of neurons are responsible for synthesizing and secreting the decapeptide gonadotropin-releasing hormone (GnRH) [24]. GnRH is released from the nerve terminals at the median eminence as timed, highly synchronized secretory bursts [4, 25, 26] and is released into the hypothalamic-hypopheseal portal system, where it is carried to the adenohypophysis of the pituitary [2, 20, 27]. GnRH stimulates gonadotrope cells of the anterior pituitary expressing the GnRH receptor, GnRH-R to synthesize and secrete pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) into portal circulation in a pulsatile fashion [2, 28, 29]. As trophic effectors, LH and FSH take part in initiating gametogenesis at the time of puberty at the level of the gonads, as well as result in sexsteroid production in the gonads, leading to alterations of other hormone and peptide levels, in a developmental- or stage-specific manner [30, 31]. Furthermore, gonadal steroid hormones mediate an additional level of regulation as they are involved in extensive feedback mechanisms at both the level of the hypothalamus and the pituitary, further regulating GnRH and gonadotropin secretion [4, 31, 32] (Figure 1.1). 3

16 Upstream neuronal afferent networks (sex steroids, metabolic signals, stress, neurotransmitters, seasonal cues) Hypothalamus POA/ME GnRH neuron GnRH Anterior Pituitary LH, FSH Testes/Ovaries Sex steroids Figure 1.1. A schematic of the mammalian hypothalamic-pituitary-gonadal (HPG) axis. The hypothalamus is responsible for integrating a variety of different inputs, both central and peripheral, including neuropeptidergic, hormonal, metabolic, and environmental. Integration of these inputs are reflected in alterations in GnRH neuronal activity. GnRH is secreted in a pulsatile manner into the hypophyseal portal system, which vascularizes the anterior pituitary. Here, GnRH acts on its GPCR, GnRH-R, which is expressed on the gonadotropes of the pituitary, stimulating release of the gonadotropic hormones, LH and FSH into circulation. LH and FSH act on the gonads, in order to facilitate gonadal development and maintenance, as well as gametogenesis, and steroidogensis. The gonadal steroids, namely estrogens and testosterones, then participate in negative feedback mechanisms at all levels of the HPG axis, regulating hormonal levels. 4

17 1.2.1 Gonadotropin-releasing Hormone (GnRH) Neurons: GnRH, also known as luteinizing-hormone-releasing hormone (LHRH) was initially discovered in mammals by the independent laboratories of Andrew Schally and Roger Guillemin [33, 34]. The developmental pattern followed by GnRH neurons is precisely orchestrated, whereby neurons originate in the olfactory placode, migrate along the vomeronasal nerves to cross the cribiform plate, entering the medial forebrain and eventually the hypothalamus [35-38]. The GnRH neuronal population is comprised of a small heterogeneous population of neurons situated in the medial preoptic area (mpoa) of the mammalian hypothalamus [2, 39, 40]. With the use of retrograde tracing and immunochemical staining studies, it has been demonstrated that nearly 70% of GnRH neurons in the mpoa extend towards the neurosecretory zone of the median eminence (ME) at the base of the hypothalamus, which signifies an important boundary between neuronal pathways and endocrine function [41]. An additional smaller population of GnRH neuron terminals exists near the organum vasculosum of the lamina terminalis (OVLT), which is beyond the blood-brain-barrier [42-44], and therefore suggests an intricate role of GnRH neurons in integrating an array of physiological cues. It has been demonstrated that the failure of GnRH neuronal migration and appropriate network development, results in hypogonadotropic hypogonadism (HH), a condition commonly associated with low gonadotropin and sex steroid levels, as well as the delayed or absence of sexual maturity all of which is attributable to the underlying lack of pulsatile GnRH secretion [45]. Most often, HH is implicated with mutations to genes that are involved in migration, secretion, and action of GnRH neurons at the pituitary. For example, HH often presents in the genetic disorder Kallman syndrome, where mutations can occur in genes encoding proteins that are involved in GnRH neuronal migration such as KAL1 [36, 46]. 1.3 Regulators of HPG axis and GnRH neurons 5

18 The fluctuating pulse frequency of GnRH release is the chief mechanism by which the body is able to modify its reproductive status during maturation and developmental processes such as, puberty and cyclic changes in adulthood [29, 47-49]. To date, a range of HPG axis modulators have been identified, which include gonadal steroids, neurotransmitter and neuropeptide systems, metabolic signals, and environmental cues. The sex steroids produced by the gonads in males and females operate as a crucial regulatory component of the HPG axis, possessing trophic and regulatory signals in a range of target tissues, including the CNS. Three main classes of sex steroids exist including, estrogens, androgens, and progestogens. Acting at the level of the hypothalamus, and more specifically, directly or indirectly at the GnRH neurons, these hormones exert their actions via stimulatory and/or inhibitory mechanisms [50] Estrogens Estrogen plays a pivotal role at every level of the reproductive axis in both males and females, controlling GnRH and gonadotrophin secretion via regulatory feedback loops. Along with the other sex steroids, estrogens play a prominent role in reproduction, and the sexual differentiation of peripheral and central brain tissue organization [51, 52], as well as energy homeostasis, neuronal growth and differentiation, mood, and cognition [53, 54]. Three important estrogens are found in the human body which are all derived fromthe common C-19 androgen precursors,,and they include: 1) estrone (E1); 2) estradiol (E2), which comes in two subforms (17β-estradiol and 17α-estradiol); and 3) estriol (E3), [55]. Estradiol is recognized as the most potent estrogen produced by the ovaries in females of reproductive capacity [56]. More specifically, 17β-estradiol is considered to be the most potent and predominant form of estrogen, and is most often used for in vivo and in vitro experiments. The bimodal effect of E2 on the hypothalamus is well established, whereby it exerts positive and negative feedback mechanisms on the GnRH neuronal network [57]. Numerous in vivo and in vitro studies have demonstrated that basal E2 levels inhibit GnRH synthesis and 6

19 secretion throughout the majority of the female cycle, acting in a negative feedback manner [58-62]. On the other hand, during the late follicular phase, as E2 levels increase, a stimulatory feedback system is elicited, increasing the GnRH pulse frequency and secretion, considered the GnRH surge [63-65]. Although this fluctuating pattern of E2 signaling as a regulator of GnRH is generally accepted, much controversy surrounds the mechanisms through which this occurs. E2- responsive GnRH neurons were reported in initial studies conducted in the guinea pig [66]; however, later reports found minimal or no co-expression of GnRH and the estrogen receptor (ER α) in mammalian GnRH neurons in vivo [67-69], leading to the proposal that GnRH is regulated by E2 exclusively via innervating afferent neurons. However, following the discovery of another estrogen receptor, ERβ, this view was challenged, as GnRH neurons in vivo were found to express ERβ with the use of immunocytochemical studies [70, 71]. Complementing this finding, the GT1-7 neuronal model demonstrates expression of both ERα and ERβ genes [59], and mrna expression for these receptors was found in both prepubertal and adult GnRH neurons using single cell RT-PCR [72]. Additionally, in humans 10-20% of GnRH neurons contain nuclear ERβ immunoreactivity, and a functional estrogen response element (ERE) has been identified within the human GnRH gene, implying a putative direct ER-mediated mechanism [57, 73]. Overall, these findings suggest that GnRH neurons are in fact directly regulated by E2, though the exact signaling mechanisms remain unknown Estrogen receptors Three estrogen receptors are known to mediate the biological actions of estrogens, participating in numerous aspects of cellular and reproductive physiology. Two of these receptors, estrogen receptor-α (ERα) and estrogen receptor-β (ERβ), are transcript products of two separate genes and therefore exist as two distinct protein forms [74]. ERα and ERβ are members of the nuclear receptor subfamily with gene structures characteristic to that of the 7

20 steroid receptor superfamily: possessing: an N-terminal domain involved in transcriptional activation; a highly conserved DNA-binding domain (DBD); the C terminal domain containing the ligand binding domain (LBD); and the hinge region linking the DBD to the LBD [75]. Furthermore, ERα and ERβ share a relatively high sequence homology: 90% homology in the DBD and 53% homology in the LBD [74, 76]. While the existence of these two receptors does not always overlap, depending on the cell type, they may exist as functional homodimers or heterodimers [74, 77, 78]. Co-expression of ERs is found within the epididymis, mammary glands, adrenal glands, thyroid, and throughout the brain [74, 79]. Nonetheless, in most target organs, one receptor type pre-dominates [80, 81], and this is dependent on the specific cell type. The ligand-dependent classical signaling mechanism for these receptors follows that upon binding of E2 to ER, the receptor acts as a transcription factor (TF) to regulate genes containing an estrogen response element (ERE), a cis-acting enhancer element, located within the regulatory region of the target gene [79]. Additionally, it is now accepted that an ER may bind to an ERE in the nucleus to signal functionally upon activation by extracellular signals, such as growth factors, in the absence of E2. This is therefore still considered ligand-independent signaling mechanism, and depends on phosphorylation of the ER by cellular kinases. Furthermore, studies report induction of genes lacking an ERE sequence by E2 bound to ER. For example, this tethering mechanism is supported by the findings that ERα can activate IGF-1 and collagenase expression, mediated through interactions with receptors for Fos and Jun at an AP-1 binding site on the target gene [82], thereby mediating ERE-independent signaling. Interestingly, ERα and ERβ have also been located at the membrane, facilitating non-genomic, rapid biological effects of estrogen signaling in bone, breast, vasculature and the CNS. Hence, cross-talk exists between ER and membrane-coupled tyrosine kinase pathways, where E2 can also active MAPK signaling pathways in specific cell types [83, 84]. A membrane-bound estrogen receptor, Gpr30, has only recently been identified [85]. As a member of the G protein-coupled receptor (GPCR) family, Gpr30 has seven transmembrane 8

21 domains and belongs to the class A rhodopsin-like receptors, more specifically, the chemokine receptor-like 2 subfamily [85-87]. Gpr30 expression has been reported in the ovary, heart, lung, liver, and throughout the brain [87-89]. As a membrane-bound receptor, the mechanisms of action subsequent to Gpr30 activation are very different from those following ERα and ERβ activation, however, the biological activities of all ER-mediated signaling may overlap or be complementary [74]. E2 is capable of activating gene transcription from both pools of membrane-bound and nuclear receptors, whereby membrane receptors may induce protein signaling to transiently activate gene transcription, which can be further sustained by actions of the nuclear receptors [74]. On the other hand, signaling from the membrane may act to amplify the actions coming from the nuclear receptors [74]. Together, ERα, ERβ and Gpr30 are recognized as endogenous estrogen receptors and have demonstrated expression and activity in the hypothalamus, specifically, at GnRH neurons, where they mediate an array of steroidal-mediated processes Other Steroid Hormones Testosterone is an important androgen, produced primarily in the Leydig cells of the testis in males, with small amounts being produced in the ovaries of females [90]. Following its synthesis, testosterone is converted into dihydrotestosterone (DHT) in androgen-sensitive cells, containing the enzyme 5α-reductase type 2 [91]. DHT and similar androgens function at the level of the hypothalamus, restricting GnRH secretion, as well as, at the pituitary to modulate LH and FSH secretion [92, 93]. Presently, controversy surrounds the mechanisms by which androgens influence GnRH, due to the fact that the presence of an androgen receptor (AR) on GnRH neurons has not been supported in vivo to date. However, in vitro models have successfully demonstrated co-expression of functional AR and 5α-reductase type 2 enzyme with GnRH neurons [91, 94, 95]. Furthermore, GnRH expression at the level of the GnRH 5 regulatory region is induced following DHT treatment [94, 95], suggesting that androgens are in fact 9

22 regulators of GnRH synthesis, though more in vivo studies are required to better elucidate the mechanisms of signaling. Progesterone (P) is another critical steroid hormone that is secreted by the corpus luteum following ovulation in females, acting under the influence of GnRH and LH hormones [96]. Pregnolone is converted to P via the enzyme CYP11A1; however, LH- stimulated de novo synthesis of progesterone also occurs in the endometrium and CNS [96, 97]. P is necessary to induce the transcription of specific genes in the endometrium that are involved in successful implantation of the blastocyst. Secondly, it is responsible for modulating GnRH and LH secretion at the hypothalamic-pituitary level by suppressing GnRH pulse frequency, which in turn enriches FSH in the pituitary gonadotrope cells and prevents a second LH surge [96]. Suppression of GnRH and LH levels by P is dependent on previous estrogen exposure Using a female ovine model, Skinner and colleagues demonstrated that following long-term estradiol deprivation, the effects of progesterone on LH secretion were lost [98]. Furthermore, they demonstrated that the inhibitory actions of P on GnRH are mediated through an estrogen-dependent nuclear progesterone-receptor system [98]. Several in vivo studies have further shown that the P-mediated suppression of GnRH neurosecretion is maintained in the presence of GABA and glutamate AMPA/NMDA receptor antagonists, suggesting that P acts independently of neuropeptidergic and neurotransmitter inputs [98, 99] Glucocorticoid hormones An organism s exposure to stress, either acute or chronic, generally results in suppression of the HPG axis, compromising reproductive status [ ]. Stressors have been demonstrated to activate the hypothalamic-pituitary-adrenal (HPA) axis, which involves the release of corticotropin-releasing hormone (CRH) from the hypothalamus, and adrenocorticotropic hormone (ACTH) release from the pituitary. ACTH stimulates glucocorticoid (GC) release from the adrenal gland, acting wherever the glucocorticoid receptor (GR) is expressed. In both GnRH- 10

23 secreting cell lines and rodent models, GCs have demonstrated a direct suppressive role on hypothalamic GnRH neurons expressing GR, inhibiting both GnRH biosynthesis and release [ ]. Similarly, at the level of the pituitary GCs suppress the synthesis and secretion of the gonadotropic hormones [105] Neurotransmitters Several neurotransmitter populations have been shown to make synaptic connections with GnRH neurons and further demonstrate the ability to regulate GnRH gene transcription, protein synthesis, and peptide secretion into the hypophyseal portal system. A great number of different innervating neuronal systems are implicated in modulating the behavior of the GnRH neurons and have emerged as having an elementary role in activation of GnRH neurons at puberty [ ]. GnRH neuronal expression of the receptors for innervating neuronal systems, such as GABA and glutamate, have shown to vary in their coexpression profiles throughout the reproductive lifecycle [ ], thereby altering the sensitivity and responsiveness of the GnRH neurons. Together, the excitatory and inhibitory afferent neuronal systems have demonstrated to be crucial components for the generation of synchronized activation of pulsatile GnRH secretion, necessary for the maintenance of reproductive capacity [116, 117]. The presence of amino acid neurotransmitter GABA in the medial hypothalamus has been confirmed with various methods, including immunocytochemistry [118] and use of specific GABA antibodies [ ]. It is recognized as the predominant inhibitory neurotransmitter within the CNS [122]. GABA expressing neurons contact GnRH neurons expressing the GABA receptor in the hypothalamus [123, 124] and inhibit GnRH secretion [124, 125], as well as, suppress gonadotrophin secretion from the pituitary [126]. In addition, it has been shown that E2 feedback is relayed to GnRH neurons via GABA in the mpoa, whereby E2 generated a 50% increase in GABA release in the mpoa, resulting in a reduction in LH secretion [1, 127, 128]. 11

24 Furthermore, in mouse, rat, and sheep, GABA concentrations at the GnRH cell body decrease just prior to the pre-ovulatory GnRH surge coinciding with maximal estrogen levels [ ]. Glutamate is the excitatory neurotransmitter opposing GABAergic actions throughout the CNS [ ] and is also a key regulator of GnRH neurons [133]. Using immunohistochemical and in situ hybridization studies, identification of the glutamate receptor in the hypothalamus has been localized to several nuclei, including mpoa, ARC, VMH, and SCN [134, 135]. Furthermore, glutamatergic fibers in the hypothalamus are in close proximity to GnRH perikarya in the POA and GnRH axons in the ME [112, 136]. Analysis of GnRH neurons has demonstrated expression of all three ionotropic glutamate receptors, NMDA, AMPA and kainite [111, 114, ]. In addition, the receptors are expressed in several hypothalamic nuclei associated in the secretion of GnRH, including AVPV, ARC and ME [139, 140]. The GT1-7 cell line represents an immortalized GnRH cell line with expression of glutamate receptor subunits [ ]. Studies have demonstrated that incubation of GT1 cells with either AMPA, kainite, NMDA or glutamate produces a rapid NMDA-mediated increase in GnRH mrna levels [138], augmented GnRH release, in addition to increased intracellular calcium concentration [ ]. Furthermore, addition of NMDA receptor antagonists, MK-801 or AP-5, inhibited the increase in GnRH release that was previously measured following NMDA stimulation [149] RFamide peptides (GnIH and Kisspeptin) In 2000, Tsutsui and colleagues discovered a novel avian hypothalamic argenine phenalalanine (RF) amide dodecapeptide with the unique ability to inhibit gonadotropin release from the anterior pituitary, and thus it was named gonadotropin-inhibitory hormone (GnIH) [150]. When incubated with quail anterior pituitary primary culture for 100 minutes, the GnIH significantly inhibited LH release in a dose-dependent manner, and was found to have a similar effect on FSH release from the pituitary, with no effect on prolactin release [150]. Following immunohistochemical studies, GnIH has been localized to cell bodies in the periventricular 12

25 nucleus (PVN) and to nerve terminals in the ME [150], and RT-PCR analysis in conjunction with Southern blot analysis has revealed GnIH mrna in the hypothalamus [151], with highest GnIH concentration in the diencephalon [152]. The GnIH receptor has been recognized as a novel seven transmembrane G protein-coupled receptor (Gpr147), binding the GnIH peptide in a concentration-dependent manner [153]. Analysis of RT-PCR products have demonstrated Gpr147 mrna expression in the pituitary and the diencephalon [153], suggesting that GnIH acts to inhibit gonadotropin release at the pituitary [152, 153]. As well, it was shown in sparrows that in vivo GnIH treatment rapidly inhibits GnRH-induced LH release [154], suggesting that GnIH may also act at the hypothalamus to inhibit GnRH release. Using a gene database search, existence of mammalian cdna encoding a novel RF-amide peptide structurally similar to the avian GnIH was discovered [155]. The mammalian RFRP gene was found to encode a prohormone that generates two biologically active RF-amide peptides, RFRP-1 and RFRP-3 [155]. Additionally, in rat, bovine, and humans, RFRP-1 and RFRP-3 were found to inhibit camp production upon binding to Gpr147, without changing intracellular calcium concentration [155], suggesting linkage of Gpr147 to either Gi or Go proteins but not to G q/11. Gpr147 expression has been localized to the hypothalamus, pituitary and the gonads in mammals [ ]. RFRP-3 is secreted in a pulsatile fashion, and it has demonstrated actions at the pituitary directly opposing the actions of GnRH [157, 159]. RFRP-3 neurons have also been found to directly oppose GnRH neuronal cell bodies in the POA; 40% in Syrian hamsters [160], and 63-75% in the murine hypothalamus [161], leading to the general agreement that RFRP-3 acts on hypothalamic GnRH neurons in addition to acting at the level of the pituitary. Several studies have shown RFRP-3 to have a similar neuroanatomical distribution throughout the CNS as was observed in the avian species, and furthermore, appears to function as a hypophysiotropic molecule capable of modulating the mammalian reproductive axis. Due to their similarities, for the purpose of this thesis, GnIH will refer to both the avian peptide and its mammalian homologue RFRP-3. 13

26 Kisspeptin is another RFamide peptide shown to regulate GnRH neurons in the hypothalamus. Studies have now confirmed in several mammalian species, that kisspeptin administration, whether central or peripheral, produces a significant rise in plasma LH and to a lesser extent, FSH [49, ]. Furthermore, it was demonstrated that following pretreatment with a GnRH antagonist, the stimulatory effect of kisspeptin on gonadotropins was lost, suggesting that rather than acting directly at the pituitary, kisspeptin stimulates release of GnRH into the portal system, which then stimulates the pituitary to increase gonadotropin release [162]. This is supported by double label in situ hybridization studies which reveal that 60-90% of GnRH neurons in the rodent hypothalamus express the kisspeptin receptor, Gpr54 [109, 163, 165], and work with rodent hypothalamic explants show that these same GnRH neurons demonstrate c-fos induction with kisspeptin administration [109, 164]. The explants were also used to successfully demonstrate release of GnRH [167, 169], and increase in electrical activity following kisspeptin stimulation [109]. In addition however, there is evidence suggesting that kisspeptin also acts indirectly on GnRH neurons to modulate GnRH release via its actions on other neurotransmitters and neuronal systems which also are known regulators of GnRH, such as GABA, glutamate and NPY neurons [ ]. (For more information on kisspeptin see Section 1.4 below). 1.4 Kisspeptin Discovery of Kisspeptin and the Kiss-1R (Gpr54) Following identification of kisspeptins, our understanding of the physiological control of reproduction and of the pathophysiology of reproductive diseases was revolutionized. In 1996, Lee and colleagues identified the first component of the kisspeptin system, the Kiss-1 gene [14]; however, no linkage to reproductive physiology was made until several years later. The group utilized several melanoma cell lines with varying metastatic abilities for subtractive hybridization experiments, and the findings demonstrated the selective overexpression of Kiss-1 mrna in metastasis-suppressed tumour cells [14]. Investigators at the Pennsylvania State College of 14

27 KiSS1 gene Transcription KiSS1 mrna Kisspeptin-145 NH Translation COOH Signal Peptide Kisspeptin Kp Kp RF-NH2 121 RF-NH2 121 RF-NH2 Kp RF-NH2 Figure 1.2. Schematic illustration of the proteolytic processing of Kiss-1 gene to generate active kisspeptin peptides. The initial peptide product of the Kiss-1 gene is a 145 amino acid preprohormone (Kp-145) which is biologically inactive. Prohormone convertases cleave Kp-145 into kisspeptin-54 (Kp-54), which is amidated at the C terminus and considered to be the major product of the Kiss-1 gene (also known as Metastin). Smaller kisspeptins have been identified, Kp-14, Kp-13 and Kp-10, all of which have the same 10 AA sequence conserved at the C terminus, and are capable of activating Gpr54 upon binding. 15

28 Medicine in Hershey, Pennsylvania, discovered the Kiss-1 gene, and wanting to associate the discovery with their hometown and its most famous product the Hershey chocolate Kiss the group named the gene Kiss-1 [14, 15]. Due to its anti-metastatic properties in tumour cells, the peptide was originally named metastin and in the years that followed, studies on the Kiss-1 gene were focused in cancer biology [14, 174, 175]. Radiation hybrid mapping and FISH analysis localized the Kiss-1 gene to the long arm of the human chromosome 1q32 [176]. The gene encoding the kisspeptin receptor, Gpr54 or Kiss1R, was first isolated in 1999 as a cdna clone, encoding a novel GPCR [15]. The group performed a PCR search of rat brain cdna and obtained a clone with an open reading frame (ORF) encoding a 398 amino acid long receptor, sharing significant sequence homologies in the transmembrane regions with the rat galanin receptors GalR1 (45%), GalR3 (45%) and GalR2 (44%) [15]. Despite the overlapping expression patterns with the galanin receptor, transfection studies of COS-7 cells with vectors containing Gpr54 cdna were performed and results revealed absence of any specific binding with 125 I-human galanin, although indicated that the endogenous ligand of Gpr54, like galanin, was peptidergic in nature [15]. The Gpr54 gene was subsequently mapped to chromosome 19p13.3, containing 4 introns and 5 exons [15-17]. Due to the molecular weight of 75 kda, which is larger than would be predicted, it has been proposed that Gpr54 is post-translationally modified by mechanisms of glycolysation at the N-terminus, or by palmitoylation at the C-terminus to generate the functional receptor [15, 16]. The peptide product of the Kiss-1 gene was identified in 2001 as the endogenous ligand to the previously orphaned GPCR, Gpr54, and the resulting peptides were renamed kisspeptins a group of peptides sharing structural similarities and deriving from a common precursor [16, 17, 177]. The initial product of the Kiss-1 gene is a 145 amino acid precursor protein, Kisspeptin-145, containing a 19-amino acid signal sequence, as well as potential sites for cleavage (dibasic residues at amino acids 57 and 67), termination, and amidation (at amino acids ) [16, 16

29 17, 177]. Distinct kisspeptins are derived from differential processing of the precursor kisspeptin- 145 [16]. The major product of the Kiss-1 gene, a 54 amino acid amidated peptide, is kisspeptin- 54 (kp-54), which can be further truncated into C-terminal fragments, kisspeptin-14, -13 and -10 [17]. These smaller kisspeptins are thought to be degradation products of the unstable kisspeptin- 54 peptide, as no obvious cleavage sites have been identified that would generate these smaller peptides [177, 178]. All four kisspeptins share a common Arg-Phe-NH2 motif at the C-terminus, grouping them as part of the family of peptides called RFamide related peptides (RFRPs) [179]. Additionally, all four kisspeptins possess similar efficacy and binding affinity to the kisspeptin receptor in vitro, indicating the significance of the conserved C-terminal for binding and subsequent receptor activation [177] (Figure 1.2). Kisspeptins were not linked to reproductive physiology until 2003, when two unrelated groups utilized mapping of two consanguineous pedigrees and reported that loss-of-function mutations in the kisspeptin receptor, Gpr54, caused patients to present with a failure to progress through puberty, as well as, HH [10, 180]. This finding was further substantiated by rodent studies, where mice with targeted deletions of Gpr54 presented with reproductive dysfunction, the only phenotypic anomaly linked to receptor mutation [180, 181]. Specifically, male mice presented with small testes, and females presented with a delay in vaginal opening and absence of follicular maturation [180]. Furthermore, both males and females maintained responsiveness to GnRH and exogenous gonadotropins, as well as, showed typically normal hypothalamic GnRH levels [180]. Several years later, Kiss-1-null mice were generated and presented with a phenotype identical to the previous Gpr-54 KO animals: low gonadotropins and sex steroids, small gonads, immature sperm and ovaries in males and females, respectively. Nonetheless, completely normal GnRH neuronal migration and appropriate HPG axis activation was seen following exogenous kisspeptin administration [182]. These results were the first of many to highlight the importance of kisspeptin and its receptor in reproductive physiology, as being elemental in the initiation of 17

30 gonadotropin secretion at puberty and maintenance of reproduction functioning for life, which cannot be overcome by compensatory mechanisms Kisspeptin and Gpr54 expression Kisspeptin peptides are highly conserved and their expression has been identified in many mammalian and non-mammalian vertebrates, both centrally and peripherally. Kisspeptin expression has been studied in several species, including mouse [30, 109, 162, 183, 184], rat [163, 185, 186], monkey [49, 187, 188], sheep [ ], goat [194, 195], and most recently humans [188, 196]. The most abundant information has been supplied by studies performed in rodents, and for this reason, the following expression analysis refers to that obtained from rodent work. High levels of Kiss-1 mrna and protein have been identified in hypothalamic nuclei including the arcuate (ARC) nucleus, anteroventral periventricular (AVPV) nucleus and periventricular nucleus (PVN), with lower expression levels in found in the anterodorsal POA [162, 183, 184, 197], supporting the idea of kisspeptin as a player in the HPG axis. Kisspeptin mrna and protein expression have also been observed in other regions of the CNS beyond the hypothalamus, including medial amygdala, the bed nucleus of the stria terminalis, subfornical organ, paraventricular thalamus, periaqueductal grey and locus coerulus [86, 162, 183]. Furthermore, identification of kisspeptin mrna and protein has been found in peripheral organs and tissues relating to reproductive function, including the placenta, cells of the corpus luteum, interstitial gland, the testes, thecal layer of the ovary, umbilical vein, as well as non-reproductive organs including pancreatic islet cells, kidney, liver, small intestine and coronary artery [14, 17, 86, 162, 183, ]. Interestingly, the typically low plasma levels of kisspeptin have been shown to rise substantially during the 8 th week of pregnancy [201], potentially playing a role in mediating the suppression of GnRH release from the hypothalamus and inducing quiescence of the HPG axis [202, 203]. Furthermore, since the majority of studies are focused on the hypothalamic kisspeptin system, the function of kisspeptin and Gpr54 in other brain regions, and 18

31 in the periphery remain largely unknown. CNS tissue distribution of Gpr54 demonstrates a complex pattern, showing highest expression in the medulla pons, midbrain, hippocampus, cortex, frontal cortex, and striatum. Interestingly, this unique expression profile resembles that of galanin receptor [15]. In situ hybridization of rat brain sections, revealed greatest expression of Gpr54 in hypothalamic and amygdaloid nuclei [15]. In the periphery, Gpr54 mrna was previously localized to the liver and intestine [15]. However, a more recent study using RT-PCR and various human tissues, found Gpr54 transcripts abundant in the placenta, pituitary, spinal cord and pancreas, with lower expression levels in several different regions of the brain, stomach, small intestine, thymus, lung, testes, kidney and fetal liver [177] Kisspeptin neurons in the ARC and AVPV nuclei The two major populations of kisspeptin-expressing neurons were initially identified in the rodent hypothalamus, and subsequently identified similarly in several different mammalian species. The first population of kisspeptin neurons in the hypothalamus was localized to the ARC nucleus, or the equivalent region in primates, the infundibular nucleus [30, 49, 162, 183, 188, 190, 194, 196, 197, 204]. The second major population of kisspeptin neurons has been localized to the preoptic area (POA), which is at the rostral periventricular area of the third ventricle (RP3V) in rodents, also known as the AVPV nucleus [30, 162, 183, 184, 197]. Immunocytochemistry studies have demonstrated that both of these populations of kisspeptin neurons project their axon fibers to GnRH neurons in the mpoa and ME of the hypothalamus, and this finding has been reported in mice [30, 205, 206], sheep [193], primates [187] and humans [196]. More recently, electron microscopy studies have revealed direct contact between kisspeptin fibers and GnRH neurons in mice [206]. While both populations of kisspeptin neurons express estrogen receptors and show direct regulatory responses to E2 stimulation, they are differentially regulated, and thus are thought to mediate different physiological modes of GnRH 19

32 secretion. (See Section 1.5 for more information on estrogen as a regulator of kisspeptin neurons) ARC KNDy neurons An accumulation of evidence has supported the existence of anatomical heterogeneity among kisspeptin neuronal populations, most notable in the ARC population where many neurons expressing Kiss-1 also express Neurokinin B (NKB) and dynorphin (Dyn), resulting in their naming as KNDy neurons [189, 192, ]. The co-localization of KNDy peptides observed in the ARC is unique among all other brain regions and hence is conserved across several mammalian species including the rat [210], mouse [211], sheep [192], and goat [195]. Interestingly, KNDy cells form reciprocal interconnections with one another (KNDy-KNDy cell connections), in addition to their projections to GnRH neurons [210, 212, 213]. Interestingly, while kisspeptin and NKB have stimulatory effects on GnRH neurons, Dyn is inhibitory [211, 214]. Together, the combination of excitatory and inhibitory signals within this network has developed the foundation for models implicating KNDy ARC neurons in the generation and control of pulsatile GnRH secretion [6, 195, 208, 211, 214] AVPV kisspeptin neurons Similar to ARC kisspeptin neurons, those found in the AVPV also have a unique coexpression profile with other neuropeptides, which include galanin and Met-enkephalin [206, 215]. Studies conducted in ovariectomized (OVX) mice treated with estradiol, demonstrate that in OVX female mice treated with estrogen (OVX+E2), 87% of AVPV kisspeptin neurons were galanin positive, while only 12% of ARC kisspeptin neurons stained positive. Furthermore, in the OVX+E2 mice, kisspeptin afferents to GnRH neurons showed galanin-immunoreactivity with an incidence of 22.5%, which was significantly greater than the 5.7% incidence measured in the OVX mice not receiving E2 treatment [206]. The physiological significance of this coexpression 20

33 Kisspeptin neuron ARC Hypothalamus (POA/ME) GnRH neuron Kp Kp Kisspeptin neuron AVPV GnRH Anterior Pituitary LH, FSH Testes/Ovaries Sex steroids Figure 1.3 Schematic summarizing the current mechanism through which kisspeptin neurons are regulated by estrogen in the HPG axis. This working model suggests that estradiol is involved in the direct regulation of kisspeptin-expressing hypothalamic neurons in the arcuate (ARC) nucleus and anteroventral periventricular (AVPV) nucleus, both of which express the estrogen receptors (ER). Furthermore, the two population of kisspeptin neurons are differentially regulated by estrogen, where ARC kisspeptin neurons are inhibited by estrogen, AVPV kisspeptin neurons are stimulated. Thus the two populations mediate two different modes of GnRH secretion: ARC kisspeptin neurons facilitate pulsatile GnRH secretion which governs majority of the estrous cycle, while AVPV kisspeptin neurons are responsible for generating the GnRH surge which induces the LH surge and ovulation. 21

34 remains unknown; however, these findings are indicative that AVPV neurons innervating GnRH neurons express galanin in an estrogen-dependent manner, perhaps providing an additional dimension to kisspeptin-mediated GnRH regulation Signaling pathways activated by kisspeptin The primary studies delineating the functional characteristics of the kisspeptin-gpr54 signaling pathways were performed by Kotani and colleagues and utilized CHO K1 cells expressing Gpr54 [177]. Binding and functional assays indicated that kisspeptin-54, -14, -13 and -10 possess equal affinity and activity at the receptor, suggesting the necessity of the conserved C-terminal region of the peptide for binding and activation of Gpr54. Kp-10 was found to bind in nanomolar affinities to rat and human Gpr54 expressed in the heterologous cell line used, activating phospholipase C (PLC) and PIP2 hydrolysis. This is accompanied by accumulation of inositol (1,4,5)-triphosphate (IP3), Ca 2+ mobilization, arachidonic acid release, activation of phosphokinase C (PKC), as well as, ERK1/2 and p38 MAP kinase phosphorylation [177] (Figure 1.4). A similar study by Muir and colleagues used HEK293 cells to study the signaling pathways activated by kisspeptin stimulation [16]. Results demonstrated Ca 2+ mobilization, additionally; kisspeptin had no effect on either basal or forsaklin-elevated levels of intracellular camp, suggesting that Gpr54 is coupled to G proteins of the G q/11 subfamily rather than those of the G s and G i/o subfamilies [16]. It has become apparent that the above-mentioned set of protein kinases involved in kisspeptin-induced Gpr54 activation is strongly dependent on the cellular context studied. For instance, anaplastic thyroid cancer (ARO) cells, expressing Gpr54 demonstrated kisspeptin activation of ERK1/2, but not of p38 or PI3K/Akt mitogen activated protein kinase (MAPK) pathways [216]. On the other hand, in a cell model derived from Gpr54-null thyroid cancer cells 22

35 with stable overexpression of Gpr54, kisspeptin stimulated both ERK1/2 and PI3K/Akt pathways [217]. Finally, studies using two pancreatic cancer cell lines with endogenous Gpr54 expression both demonstrated kisspeptin stimulation of ERK1/2; however, activation of p38 pathway occurred in only one of the cell lines [218]. It is apparent that ERK1/2 activation is the most conserved kinase signal among the cell types studied, however its exact role in the anti-metastatic and anti-migratory actions of kisspeptin/ Gpr54 have yet to be determined. Similarly, the precise mechanisms, which are responsible for this cell-specific signaling regulation and its biological relevance, remain unknown. The ability of kisspeptin to activate the PLC-Ca 2+ signaling route in a more physiological context has been analyzed by Castellano and colleagues, who evaluated the contribution of different second messengers to kisspeptin-induced GnRH release from hypothalamic tissue cultured ex vivo, by utilizing selective blockers of different second messengers [198]. Blockade of PLC or depletion of intracellular Ca 2+ from internal stores completely eliminated kisspeptinelicited GnRH release, while inhibition of adenylate cyclase activity or blockade of extracellular Ca 2+ influx did not alter the ability of kisspeptin to induce GnRH release from hypothalamic tissue [198]. These findings indicate that the ability of the kisspeptin/gpr54 system to stimulate GnRH secretion from GnRH neurons is dependent on PLC activation and intracellular Ca 2+ mobilization. Similarly, in pituitary cells expressing Gpr54 isolated from peripubertal male and female rats, it was shown that kisspeptin increases intracellular Ca 2+, increasing LH secretion [219] Kisspeptin activity in the hypothalamus Over the past decade, evidence has emerged supporting the hypothesis that kisspeptin acts on GnRH neurons directly in the hypothalamus [200], as expression of Gpr54 on GnRH neurons has been confirmed [109, 163, 165]. Further, kisspeptin immunoreactive fibers are in close association with GnRH neurons in the hypothalamus [162, 193, 204]. In vitro analysis has 23

36 Kisspeptin Kinase Cascades Gpr54 G q/11 γ α β PLC PIP 2 DAG PKC IP 3 PI3K p38 ERK1/2 ER Akt Ca 2+ Gene Transcription Figure 1.4. Schematic illustration of the signal transduction pathways activated following kisspeptin/gpr54 binding. Kisspeptin binds to the G protein-coupled receptor, Gpr54, at the membrane to activate a Gq/11 protein intracellularly. This activates phospholipase C (PLC), resulting in inositol-(1,4,5)-triphosphate (IP3)-mediated intracellular calcium release, as well as diacyglycerol (DAG)-mediated activation of protein kinase C (PKC). Furthermore, kisspeptin- Gpr54 binding can activate mitogen-activated protein kinase (MAPK) pathways, including extracellular signal-related kinase ½ (ERK1/2), p38, and PI3/Akt. 24

37 demonstrated the ability of kisspeptin to directly depolarize GnRH neurons, increasing GnRH neuronal firing rates [132, 171, ]. More recent findings support indirect actions of kisspeptin on GnRH neurons as well, acting through intermediary neuronal populations such as GABAergic cells and NPY neurons [170, 171] Kisspeptin activity in the pituitary Interestingly, kisspeptins have been identified in the ovine hypophyseal portal blood (Smith 2008), leading to the proposition that kisspeptin may act at the level of the pituitary to directly induce LH secretion from the gonadotropes. Supporting this claim, dual fluorescence labeling with a mouse monoclonal antibody identified Kiss-1 and Gpr54 expression in the pituitary gonadotrophs [223]. Further quantitative RT-PCR and immunohistochemistry studies have shown that mrna expression of Kiss-1 and Gpr54 in the pituitary is regulated by sex steroids in ovariectomized female rat tissues [223, 224]. In vitro studies demonstrated that when pituitary fragments are treated with kisspeptin, they respond with an increase in gonadotropin secretion [219, 225]. Despite these findings, use of a GnRH antagonist inhibits the typical kisspeptin-induced increase in LH [162, 163], indicating that the primary actions of kisspeptin on gonadoptropin secretion occur upstream of the pituitary. Functional studies focusing on the direct stimulatory effects of kisspeptin on pituitary gonadotropin secretion have yielded conflicting results and suggest that kisspeptin cannot independently prompt the LH surge. It is therefore likely that at the pituitary, kisspeptin acts synergistically with GnRH and estradiol to stimulate gonadotropin secretion. 1.5 Kisspeptin and HPG axis in reproduction Kisspeptin was linked to reproductive physiology in 2003 when humans and animals possessing loss-of-function mutations or targeted deletions of Gpr54 presented with HH and failure to progress through puberty [10, 180, 181]. These studies were essential to suggest at least 25

38 a permissive role of Kisspeptin/Gpr54 in reproductive function. However, a more stimulatory role of kisspeptin has been proposed following observation that administration of kisspeptin to prepubertal females rats induces LH secretion and ovulation [164]. Furthermore, studies accumulated over the last decade have lead to the current conviction that together the kisspeptin/gpr54 system functions as an essential gatekeeper of GnRH secretion, regulating and integrating central and peripheral inputs, sustaining a chief position in the hierarchy of the reproductive axis Kisspeptin activation at puberty Puberty is known as the transitional period in between childhood and adulthood, culminating in the development of secondary sex characteristics and achieving a state of reproductive capacity. During puberty onset, GnRH secretions change from a low-level and irregular pattern, to one that is regular and pulsatile [ ]a change that is necessary to stimulate pituitary gonadotropes to secrete gonadotropin hormones [29, 228]. Kisspeptin has become recognized as a critical element for developmental puberty as activator of GnRH neurons from their quiescent state [108, 109, 162, 164]. Semi-quantitative RT-PCR work on hypothalamic fragments from male and female rats, and in situ hybridization studies in male mice demonstrate that Kiss-1 mrna expression significantly increases across development in the AVPV nucleus [6, 109] suggesting that kisspeptin-gpr54 signaling in the rodent is amplified at puberty, capable of stimulating hypothalamic GnRH neurons. Using immunocytochemistry, it was found that the increase in the number of Kiss-1 cells in the AVPV of pre-pubertal animals is estradiol-dependent [197]. They then proposed that increased Kiss-1 neuronal expression generates an increase in GnRH release, ultimately enhancing estradiol production at the ovaries to generate a feed-forward mechanism, where kisspeptin AVPV neurons act as estradiol-dependent amplifiers of GnRH neuronal activity [197]. Kisspeptin has also demonstrated a triggering role in puberty onset in primates, where central kisspeptin injections stimulate an increase in LH in pre-pubertal, 26

39 agonadal male monkeys [49], suggesting that the stimulatory actions of kisspeptin can override the lack of GnRH secretion. Another study used RT-PCR to measure changes in expression of hypothalamic Kiss-1 and Gpr54 at the time of puberty in agonadal male monkeys, and in ovaryintact females. In both male and female monkeys, they reported greater hypothalamic Kiss-1 expression in pubertal animals compared to juvenile ones, while Gpr54 expression did not differ [49]. In addition to enhanced Kiss-1 expression, electrophysiological studies performed on brain slice preparations from GnRH-GFP transgenic mice have demonstrated a heightened response of GnRH neurons to kisspeptin stimulation at puberty onset [109]. While it has become evident that the changes in the kisspeptin-gpr54 system participate in the awakening of GnRH neurons at puberty to facilitate GnRH secretion, the essential trigger for these changes has yet to be elucidated Kisspeptin and the menstrual cycle Kisspeptin is currently the most powerful stimulus known for GnRH-induced LH secretion across mammalian species [162, 165]. Studies in men receiving exogenous kisspeptin treatment have illustrated that the GnRH neuronal network is capable of sensing exogenous kisspeptin administration, resetting the GnRH pulse generator and adjusting succeeding endogenous GnRH/LH pulses accordingly [168, ]. Expanding these findings to encompass regulation of GnRH by kisspeptin in females is complicated by our knowledge of Kiss-1 and GnRH regulation by gonadal hormones, which vary across the menstrual cycle. In a study by Chan and colleagues, responses to kisspeptin were studied in healthy women during the 1) early follicular phase, 2) the preovulatory period, and 3) the mid-luteal phase, and they reported that the GnRH-induced LH response to kisspeptin was greatest during the preovulatory phase, and smallest in the follicular phase [234]. These findings are corroborated by previous studies completed in humans [231, 233], rats [235, 236] and sheep [237]. It is known that estrogen increases sensitivity of GnRH neurons to kisspeptin stimulation [109, 238, 239], and 27

40 therefore likely that without sufficient circulating estrogen, GnRH neurons are not sensitive to kisspeptin stimulation. The importance of estrogen in mediating the generation of the GnRH/LH pulse is illustrated by studies utilizing estrogen agonists during the preovulatory period to generate the LH pulse [240], and estrogen antagonists to inhibit pulse generation [241]. Furthermore, it was demonstrated that kisspeptin administration in OVX monkeys was not capable of generating the GnRH/LH pulse, however, the pulse was restored upon estradiol replacement [242], validating the necessity of estrogen in generation of LH pulses and the preovulatory LH surge. More specifically, amplitude and frequency of the LH pulses are determined by the sensitivity of GnRH neurons to kisspeptin stimulation, which is modified by steroid hormone levels. This work has expanded our knowledge of how kisspeptin stimulates GnRH pulses in varying sex steroid milieus and is essential for future usage of kisspeptin or its analogues to modify GnRH pulses in reproductively challenged individuals. 1.6 Regulation of kisspeptin by afferent signals Gonadal steroid hormones The gonads, as the terminal organs implicated in the HPG axis, are the production sites for the synthesis of gonadal steroid hormones. The gonadal steroid hormones play a fundamental role in the reproductive axis, acting as both trophic and regulatory signals in a variety of target tissues, eventuating in the modulation and control of GnRH and gonadotropin secretion [31, 63, 243]. More recently, estrogen has demonstrated direct effects on the transcriptional regulation of Kiss-1 gene in the rodent hypothalamus, having differential effects: generating up-regulation of Kiss-1 expression at the AVPV nucleus, and down-regulating at the ARC nucleus [183, 184]. Findings which support AVPV Kiss-1 neurons as positive feedback targets for estradiol and as generators of the preovulatory LH surge include: 1) immunohistochemical studies that demonstrated connections between AVPV Kiss-1 neurons and GnRH neurons [30]; 2) in female 28

41 rodents it was shown that immunoneutralization of endogenous kisspeptins in the POA abolished the LH surge [244]; and finally, 3) mice with congenital absence of Gpr54 or Kiss-1 remained anovulatory through development [199]. Furthermore, results from both male and female mice indicate that early postnatal gonadectomy produces a reduction in Kiss-1 expression measured in the AVPV by 70 90% at the time of puberty, which persists throughout adulthood [197, 205]. On the other hand, the idea of ARC kisspeptin neurons as negative feedback targets of estradiol come from studies performed in male mice, rats, hamsters and monkey following castration, where a dramatic increase in Kiss-1 mrna expression in the MBH, specifically within the ARC nucleus was reported [6, 163, 184, 245, 246]. Similarly, in the female mouse, estradiol appears to mediate negative feedback actions on kisspeptin neurons in ARC, as it was shown that Kiss-1 mrna expression here reaches nadir when estradiol levels are highest during the estrous cycle [183]. Likewise, in rodents, sheep and monkeys, OVX and the associated reduction in gonadal steroids, causes an increase in ARC Kiss-1 mrna expression, which is reversible with estradiol treatment [6, 183, 185, 188, 190, 193] Metabolic factors Reproduction is a highly energy-demanding function that is nonessential for survival, and therefore the HPG axis is rapidly shut down upon energy insufficiency or in conditions metabolic stress associated with morbid obesity [ ]. Changes in the expression of Kiss-1 reflect changes in the energy status of an organism. In pubertal male and female rats fasting up to 72 hours, the negative energy balance induced a significant decrease in hypothalamic Kiss-1 mrna expression, and a concomitant decrease in circulating LH levels [198]. Leptin, a hormone produced by adipocytes, known to act in the brain regulating feeding behaviour and energy homeostasis, has more recently been proposed as a gate of reproductive function. Leptin receptor is expressed in 40% of ARC Kiss-1 neurons [250] but has not been identified in the AVPV of rats or mice [ ]. In a study using leptin-deficient ob/ob mice, a reduction in ARC Kiss-1 29

42 mrna by 35%, and cellular Kiss-1 mrna content by 40% was measured [250]. Further, in fasting wild type animals, as leptin and Kiss-1 mrna levels decreased, so did gonadotropin secretion [254, 255]. Additionally, Grehlin, a gut-derived hormone secreted by endocrine cells of gastric mucosa and a signal for energy insufficiency, is thought to act as a negative regulator of puberty onset and gonadotropin secretion, demonstrating the ability to inhibit hypothalamic Kiss- 1 expression in female rats [256]. The orexigenic neuropeptide, neuropeptide Y (NPY), was shown to enhance hypothalamic Kiss-1 expression in both animal and cell models [257], acting as a positive regulator of the HPG axis. Despite its numerous metabolic roles, insulin has yet to demonstrate regulatory effects on Kiss-1 expression [198, 257] Circadian and seasonal changes One of the most critical factors necessary for reproductive adaptations is photoperiodic, or day length changes [ ]. Photoperiod controls the pattern of melatonin secretion from the pineal gland, helping the animal to determine the current season. It was shown that in the male Syrian hamster, following a transfer from long day to short day conditions, Kiss-1 mrna in ARC decreased, preceding reproductive quiescence [245]. Further, ablation of the pineal gland prevents the suppression of Kiss-1 mrna, suggesting a melatonin-dependent mechanism [245]. In another study, male and female Siberian hamsters held in short day conditions exhibit a reduced response to exogenous kisspeptin treatment, and show negligible AVPV Kiss-1 expression, although high expression in the ARC [261]. Using immunocytochemistry, this expression pattern demonstrated a reversal in long day conditions [261]. It is difficult to make generalization from results obtained in the hamster, since kisspeptin activity in the two species (Syrian and Siberian) is different in response to day length. Nonetheless, it is evident that reproductive quiescence induced by short day photoperiods accompanies low levels of Kiss-1. The sheep is another seasonally breeding species, which becomes reproductively active with shorter days, and quiescent with longer days. In sheep, during the long day periods, Kiss-1 expression is lower and fewer kisspeptin terminals 30

43 Hypothalamus (POA/ME) GnRH neuron Kiss neuron Gpr147 GnIH neuron? GnIH neuron GnRH Anterior Pituitary GnIH LH, FSH Testes/Ovaries Sex steroids Figure 1.5 Schematic summarizing the mechanisms through which Kisspeptin and GnIH may participate in the regulation of the HPG axis. This working model suggests that kisspeptin neurons from the ARC nucleus and AVPV nucleus send projections to contact GnRH neuronal cell body and axon terminals in the preoptic area (POA) and median eminence (ME) respectively, stimulating GnRH synthesis and secretion. GnIH inhibits gonadotropin secretion, acting on its receptor, Gpr147, at the level of the pituitary and the hypothalamus. GnIH-expressing neurons have also been found to project to AVPV, innervating kisspeptin neurons which demonstrate expression of Gpr147. Therefore GnIH may suppress GnRH neuronal activity via direct actions on kisspeptin neurons, suggesting a novel mechanism of signaling. 31

44 contact GnRH neurons [190, 191, 193]. Kisspeptin has revealed a key role in the regulation of seasonal breeding in several species; however, its precise role in seasonal breeding changes remains incomplete GnIH Kisspeptin and GnIH neurons in the rodent are known to appose GnRH neurons in the hypothalamus where Gpr54 and Gpr147 are expressed [109, 156, 163, 262, 263]. Due to the paucity of current knowledge on the regulation of GnIH and Gpr147 in the central nervous system, a recent study aimed to quantify the levels of expression of the two genes throughout the brain, and very interestingly, they reported highest expression of Gpr147 in AVPV nucleus compared to all other brain regions [158]. From this finding, it was suggested that in addition to its direct actions on GnRH neurons, GnIH might act on Gpr147 expressed by kisspeptin neurons, and indirectly regulate GnRH neurons. In female rats, GnIH immunoreactive fibers have been identified in the AVPV region of the hypothalamus [264], where during proestrous 19% of AVPV kisspeptin neurons exhibit close contact with GnIH fibers, and during diestrous, 16% of these neurons demonstrated Gpr147 expression [265]. Additionally, central administration of GnIH resulted in a dose-dependent suppression of AVPV kisspeptin neuron activity, as well as, GnRH neuronal activity (Anderson 2009). These findings support a role for GnIH in providing negative input to AVPV kisspeptin neurons, and indirectly modulating GnRH neurons (Figure 1.5.). 1.7 Immortalized hypothalamic cell models for the study of neuroendocrine function The maintenance of internal homeostasis is crucial for successful activity of many biological functions, including reproduction. The hypothalamus functions as the integrative core of the CNS, enabling the control of precisely synchronized physiologic processes by integrating various peripheral inputs, as well as, the activation of neuropeptidergic and neurotransmitter systems 32

45 [ ]. Classical in vivo approaches have been fundamental in expanding our current understanding of reproduction functions in the hypothalamus particularly, demonstrating the effects of stimuli on a specific organ and on the overall physiological state of the organism [269]. Due to the heterogeneous nature and complex circuitry of the hypothalamus, including numerous afferent and glial networks, our ability to define specific cellular events and transcriptional regulation, remains poorly understood. In vitro studies have been employed to circumvent these issues, however, non-transformed primary hypothalamic culture is difficult to maintain and has a short life span. For this reason, hypothalamic cell models have been generated for the study of more complex hypothalamic mechanisms, and can be used to study the specific effects of stimuli directly on the CNS [270]. Mellon and colleagues generated the GT1 clonal neurosecretory GnRH cell lines in 1990 [271], utilizing a targeted tumorigenesis. The simian virus 40 (SV40) T antigen (TAg) was employed and targeted to the 5 regulatory region of the GnRH gene, thus yielding specific GnRH neuron expression. Following the culturing and passaging of the tumor cells, three unique and homogenous subclones were generated, appropriately named GT1-1, GT1-3, GT1-7. These cell lines have become recognized as a highly valuable in vitro GnRH cell model, as they secrete GnRH in a pulsatile manner, express specific neuronal markers, and generate a suitable response to various hormonal stimuli [94, 145, 266, ]. Successful use of this technique has encouraged other groups to produce similar models [ ] and overall has expanded our knowledge of the impact of peripheral factors on the regulation of transcriptional and neurosecretory events mediating reproduction Immortalized adult mouse hypothalamic cell lines (mhypoa-xx) Using a novel immortalization technique, the Belsham lab successfully generated 38 immortalized, embryonic hypothalamic cell lines of mouse origin [276], as well as 33 embryonic rat, clonal, hypothalamic neuronal cell models [277]. Briefly, the murine cell models were 33

46 generated following retroviral transfection of SV40 T-Ag into primary hypothalamic cultures. The heterogeneous neuronal cultures were then serially diluted until clonal populations of immortalized embryonic murine cell lines were obtained. However, to better understand cellular mechanisms involved in mammalian reproduction in mature, fully differentiated hypothalamic neurons, the Belsham group established a novel method to immortalize neurons from adult rodents. In order for cells to be infected with SV40 T-Ag for immortalization, they must be actively dividing. Ciliary neurotrophic factor (CNTF) has demonstrated the ability to promote neuronal survival and differentiation in both the central and peripheral nervous systems [278], and trigger neurogenesis in primary hypothalamic culture [279]. Therefore, to generate immortalized adult cell lines, primary hypothalamic culture from male and female mice was treated with CNTF prior to retroviral infection with SV40 T-Ag, followed by a similar protocol used to generate the embryonic cell lines [276]. In total, over 50 unique adult mouse hypothalamic cell lines were obtained, and were named accordingly, mhypoa- clone number. The cell lines express neuronal markers characteristic of mature neurons, exhibit typical neuronal morphology, and express different processing enzymes [279]. Furthermore, they have been characterized based on expression of specific neuropeptides and receptors. These cell lines serve as an indispensible model to study the signaling cascades activated by hormonal and peptide stimulation. Further, they will enhance our understanding of the complex molecular mechanisms that mediate gene regulation, neurosecretion, and neuronal activity at the level of the hypothalamus. This thesis uses two adult hypothalamic cell lines, the mhypoa-50 and mhypoa- 55, due to their strong expression of the Kiss-1 gene, as well as expression of other genes crucial for reproductive signaling, including Gpr54, ERα and ERβ. These cell lines are characterized and described in Section Immortalized adult non clonal GnRH cell model (mhypoa-gnrh/gfp) 34

47 In order to appropriately study the direct effects of neuromodulators on hypothalamic GnRH neurons, the Belsham group generated a model of adult-derived, immortalized GnRH neurons. This model was generated using similar protocol described above, whereby primary culture from 2 month-old transgenic GnRH-GFP mice [produced by Dr. Suzanne Moenter (University of Michigan, Ann Arbor, MI) [280], available through The Jackson Laboratory, Bar Harbor, ME] were isolated. Neurons were treated with recombinant rat CNTF and infected with the construct containing cdna for the SV40 TAg, and then sorted by fluorescence activated cell sorting (FACS) using a cell sorter, following a protocol previously utilized in our lab for the generation of the mhypoa-npy/gfp cell line [170]. The cell line generated represents a mature heterogenous population of GnRH neurons and has been named the mhypoa-gnrh/gfp neurons. Characterization of this cell line has demonstrated endogenous expression of the relevant reproductive and metabolic receptors and enzymes, specific neuronal markers, as well as, neurosecretory properties (McFadden et al, manuscript submitted). Although clonal and embryonic GnRH-secreting GnRH cell models exist, this novel in vitro model of GnRH neurons will be essential, as a heterogeneous, mature adult population, and non-clonal cell line is more representative of the population of native GnRH neurons in the hypothalamus. 1.8 Study Hypothesis and Aims Fertility is a complex and highly regulated process dependent on the integration of hypothalamic neuropeptides and peripheral hormone signals by the GnRH neurons, positioned at the pinnacle of the HPG axis. Kisspeptin and Gpr54 have emerged as fundamental gatekeepers of reproduction, acting upstream of GnRH neurons. It is well established that kisspeptin neurons express estrogen receptors and estradiol-mediated regulation of these neurons is nuclei-specific. Further, subpopulations of kisspeptin neurons have been found to express Gpr54 and Gpr147, suggesting putative novel autoregulatory and inhibitory mechanisms respectively, within kisspeptin neurons. There is indisputable evidence to support that as the mammalian hormonal 35

48 environment changes, during pubertal development or throughout the female menstrual cycle, sensitivity and secretory actions of the HPG axis change as well. We use kisspeptin neurons to study the regulation of genes for the receptors of afferent modulators of kisspeptin, such as hormones and neuropeptides. This will help to better understand how the efficiency of kisspeptin signaling facilitates different physiological functions of the reproductive system. In turn, we want to elucidate the downstream effects of the kisspeptin system, on the GnRH system, which ultimately governs reproduction. The current thesis seeks to explore the general hypothesis, which will elucidate changes in the sensitivity of the kisspeptin signaling system in response to afferent modulators, such as 17β estradiol and hypothalamic neuropeptides, kisspeptin and GnIH. In turn, the effects of kisspeptin on GnRH neurons will be determined. Dissecting the direct effects of afferent signals on kisspeptin neurons was previously challenging, however, with the generation of our immortalized, clonal rodent cell lines, we are now able to complete these studies effectively. In order to test our general hypothesis, this thesis has been broken down into 3 Aims, which will be presented as one chapter of this thesis. Aim 1 is presented in Chapter 3 of this thesis and utilizes immortalized adult rodent cell lines, the mhypoa-50 and mhypoa-55, both of which demonstrate strong endogenous expression of Kiss-1, as well as, other reproductive-associated receptors. In this aim, we test the hypothesis that when stimulated by 17β-estradiol, kisspeptin-expressing neurons alter their gene expression in order to adjust their sensitivity and heighten responsiveness of the kisspeptin system to various afferent inputs. To test this hypothesis: 1) we will characterize our two kisspeptin-expressing cell lines; 2) we will then focus on the regulation of kisspeptin-expressing neurons by 17β-estradiol by examining the changes in gene expression; and 3) we will elucidate the specific receptor-mediated transcriptional mechanisms involved in the 17β-estradiol-induced changes in gene expression observed previously. 36

49 Aim 2 is presented in Chapter 4 of this thesis and has utilized the kisspeptin-expressing cell lines again, mhypoa-50 and mhypoa-55. Here we test the hypothesis that hypothalamic neuropeptides, kisspeptin and GnIH regulate kisspeptin-expressing neurons, and reveal novel mechanisms of auto-regulation and inhibition in kisspeptin neuronal populations in vitro in hypothalamic kisspeptin neuronal models. This hypothesis will be tested by: 1) first, we will establishing presence of Gpr54 and Gpr147 expression in mhypoa-50 and mhypoa-55 cell lines; 2) if these receptors are detected, we will study the regulation of kisspeptin neurons by kisspeptin and GnIH peptide treatments, by measuring changes in gene expression; and finally 3) we will elucidate the protein signaling cascades activated by Gpr54 activation, induced by kisspeptin treatment. Aim 3 of this thesis will be studied in Chapter 5 where we test the hypothesis that kisspeptin and GnIH regulate GnRH biosynthesis in the novel, adult-derived, GnRH-secreting neuronal model, mhypoa-gnrh/gfp cell line. To test this hypothesis, we will: 1) characterize the expression profile of the mhypoa-gnrh/gfp cell line in vitro, specifically looking for the expression of Gpr54 and Gpr147; 2) we will analyze the effect of kisspeptin treatment on GnRH gene transcription in our cell model; and 3) we have previously demonstrated the effects of GnIH treatment in the mhypoa-gnrh/gfp cell line (Gojska et al., unpublished data), however, here we will establish a combined effect of a kisspeptin-gnih co-treatment on GnRH gene expression in the mhypoa-gnrh/gfp cell line. 37

50 CHAPTER 2: MATERIALS AND METHODS 38

51 2.1 Cell culture and reagents mhypoa-50, mhypoa-55 and mhypoa-gnrh/gfp cells were cultured in monolayer in Dulbecco s Modified Eagles Medium (DMEM) 4.5 mg/ml glucose, supplemented with 5% fetal bovine serum (FBS) (Sigma-Aldrich, Oakville, Canada) and 1% penicillin/streptomycin (Gibco, Burlington, Canada). Neurons were maintained at 37 C with 5% CO2, a methodology previously described [276, 279]. Estrogen (E2) (Sigma-Aldrich, Oakville, Canada) was dissolved in absolute ethanol to a stock concentration and stored at -20 C prior to mrna studies. During steroid treatment, cells were serum-starved for h in phenol red-free DMEM, and treatments were performed in phenol red-free medium supplemented with 5% charcoal-stripped FBS [281]. ERαselective agonist 4,4',4''-(4-Propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT), ERβ-selective agonist 2,3-bis(4-Hydroxyphenyl)-propionitrile (DPN), ERα antagonist 1,3-Bis(4- hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1h-pyrazole dihydrochloride (MPP dihydrochloride), ERβ antagonists 4-[2-Phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin- 3-yl]phenol (PHTPP), estrogen receptor antagonist 7α,17β-[9-[(4,4,5,5,5-Pentafluoropentyl)sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17-diol (ICI ), and Gpr30 antagonist (3aS*,4R*,9bR*)-4-(6-Bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-3H-cyclopenta[c]quinolone (G- 15) were obtained from Tocris Bioscience (Missouri, USA). All substances were dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, Oakville, Canada) and stored at a stock concentration of 10 μm in a -80 C freezer, and were subsequently dissolved in water to a final concentration of 10 nm (for the selective ER-agonist treatments) and a final concentration of 1 μm (for the ER antagonist treatments). RFRP-3 peptide was purchased from Sigma-Aldrich (Oakville, Canada) was dissolved in water to a stock concentration (1 μm) and stored at -80 C prior to mrna studies. For c-fos mrna studies, cell culture medium was replaced with serumfree DMEM containing 1% penicillin/streptomycin for a minimum of 4 h prior to treatments. Kiss-10 peptide was purchased from Phoenix Pharmaceuticals (Burlingame, USA) and dissolved in water to a stock concentration (10 μm) and stored at -80 C prior to mrna studies. The G 39

52 protein β (Gβ) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, USA), and the phospho-erk1/2, phospho-akt, phospho-creb and phospho-p38 antibodies were purchased from Cell Signaling Technology (Danvers, USA). 2.2 Reverse transcriptase-polymerase chain reaction Each cell line was analyzed for the expression of specific markers by RT-PCR. Cells were grown to approximately 85-90% cell confluence, and total RNA was isolated using the guanidinium thiocyanate phenol chloroform extraction method [282]. RNA was assessed for purity and concentration was determined using a NanoDrop (Ultraspec2000c spectrophotometric, Amersham Pharmacia Biotech, USA). RNA samples were treated with Turbo DNase (Ambion) prior to amplification. Samples were amplified using a one-step RT-PCR Kit (Qiagen, Mississauga, Canada) as per the manufacturer s instructions. RT-PCR was conducted according to the following protocol: 95 C for 30 s, 60 C for 30s, and 72 C for 1min (40 cycles). A total of 200 ng of RNA template from each cell line was used for each reaction. PCR products were separated and visualized on a 2% agarose gel containing 0.5 ug/ml ethidium bromide, under ultraviolet light. Products were run alongside a 100bp ladder (Fermentas, Burlington, Canada) to determine product size. Primers were designed using Integrated DNA Technologies PrimerQuest and NCBI Primer-Blast. Amplicon sizes and annealing temperatures are listed in Table Quantitative real time PCR: Hypothalamic neuronal cell lines were grown in 60 mm culture plates to 80-85% confluence. Prior to E2, Kiss-10 and vehicle treatments in the mhypoa-50 and mhypoa-55 neurons, cell culture medium was changed to phenol red-free DMEM (HyClone), supplemented with 5% charcoal- stripped FBS [59, 283] and 1% penicillin/streptomycin (Gibco, Burlington, Canada) for a minimum of 4 h. Total cellular RNA from treated and vehicle cell culture plates was isolated using the guanidium isothiocyanate phenol chloroform extraction method at the indicated time 40

53 points. For antagonist studies, the mhypoa-50 and mhypoa-55 neurons were pretreated with 1 μm of antagonist or vehicle for 1 h prior to E2 treatment (10 nm) and RNA was isolated at h time points. mhypoa-gnrh/gfp neurons were treated with vehicle, Kiss-10 (10 nm), or Kiss- 10/GnIH co-treatment (10 nm Kiss-10 and 100 nm GnIH), and RNA was collected over a 24 h time course at the indicated time points. Subsequently, RNA concentration and purity were measured with the NanoDrop 2000c spectrophotometer. Reverse transcription was performed with 2 ug/μl of total RNA, which was treated with Turbo DNase (Ambion). Subsequently, the High Capacity cdna Reverse Transcription kit was used according to manufacturer s protocol (Applied Biosystems). Further, ng of cdna template was amplified using SYBR green PCR master mix for real-time RT-PCR containing, 0.3 SYBR green dye, 1 PCR buffer, 3 mm MgCl 2, 2 mm dntps, 1 ROX reference dye, 0.3 μm gene-specific primers, and 0.2 U of Platinum Taq DNA polymerase (Invitrogen, Burlington, Canada). The SYBR primer sequences, amplicon sizes and annealing temperatures are listed in Table 2.1. Primers used for both one-step PCR and quantitative PCR were designed using an online primer design tool, PrimerBLAST. When possible, primers were designed to flank an intron to control for DNA contamination. All PCR products were run on an agarose gel to verify the molecular size of the PCR product and sequenced (TCAG DNA Sequencing Facility; Toronto, Canada) to confirm identity. Samples were run in triplicate on the Applied Biosystems Prism 7000 real-time PCR machine. Briefly, all genes were run on the real-time PCR machine according to the following protocol conditions: 50 C for 2 min, 95 C for 10 min; 40 cycles for 15 sec at 95 C, 60 C for 1 min. Analysis of qrt- PCR data was performed using the standard curve method and normalized to histone 3a 41

54 Table 2.1 List of primers used for one-step PCR and quantitative RT-PCR Gene Name Primer Sequence Accession number Amplicon Size (bp) Annealing Temp (C ) Kiss1 Gpr54 ERα ERβ Gpr30 Gpr147 GnRH Tac2 Galanin Dynorphin Metenkephalin Substance P Tyrosine Hydroxylase c-fos Histone 3a F: AGC TGC TGC TTC TCC TCT GT R: GCA TAC CGC GAT TCC TTT T F: CAC ATC CAG ACA GTT ACC AAC TTC T R: CAC GCA GCA CAG TAG GAA AGT T F: AAT TCA ATT CTG ACA ATC GAC GCC AG R: GAA TTC GTG CTT CAA CAT TCT CCC TC F: CAG TAA CAA GGG CAT GGA AC R: GTA CAT GTC CCA CTT CTG AC F: TCA GCA GTA CGT GAT TGC CCT CTT R: AGC TGA TGT TCA CCA CCA GGA TGA F: AAC ACC CTG GTC TGC TTC ATT GTG R: TGA CGG CCA GGT TGA GGA TAA ACA F: CGT TCA CCC CTC AGG GAT CT R: CTC TTC AAT CAG ACT TTC CAG AGC T F: ATT GCT GAA AGT GCT GAG CAA GGC R: AGT GTC TGG TTG GCT GTT CCT CTT F: CAT GCC ATT GAC AAC CAC AG R: GGA TTG GCT TGA GGA GTT GG F: CTC ACC CTG ACG GTC TCT GGG CTC R: TTC CTC TGG GAC GCT GGT AAG GAG F: GCA GCT ACC GCC TGG TTC GC R: CCA TCC ACC ACT CGG GGC GT F: TCG ATG CCA ACG ATG ATC TA R: AGC CTT TAA CAG GGC CAC TT F: TTC GAG AGG GAT GGA AAT GCT R: TTG GTG ACC AGG TGG TGA CAC TTA F: CAA CGA GCC CTC CTC CGA CT R: TGC CTT CTC TGA CTG CTC ACA F: CGC TTC CAG AGT GCA GCT ATT R: ATC TTC AAA AAG GCC AAC CAG AT NM_ NM_ NM_ NM_ NM_ NM_ NM_ NM_ NM_ NM_ NM_ NM_ NM_ NM_ NM_ Primers were designed to flank an intron to control for DNA contamination whenever possible. 42

55 2.4 Western Blot Analysis mhypoa-50 and mhypoa-55 cells were grown to 90% confluence, and serum-starved for a minimum of 4 h prior to kisspeptin (10 nm) treatment or vehicle. The cells were washed with cold PBS and harvested at 5, 15, 30 and 60 min using a 1X lysis buffer (20 mm Tris HCl, 150 mm NaCl, 1 mm Na2EDTA, 1 mm EGTA, 1% Triton, 2.5 mm sodium pyrophosphate, 1 mm β- glycerophosphate, 1 mm Na3VO4, 1 mm leupeptin) (Cell Signaling Technology; Danvers, USA) supplemented with 1 mm PMSF and phosphatase inhibitor cocktail (Sigma Aldrich; Oakville, Canada). The cell lysates were centrifuged at 14,000 rpm for 10 min at 4 C and the supernatant were stored at -80 C. The protein concentration was measured using a biocinchoninic acid (BCA) protein assay kit (Pierce Biotechnology, IL). 30 μg of protein was resolved on an 8% SDS- polyacrylamide gel and transferred overnight onto immuno-blot PVDF membrane (Bio-rad Laboratories; Hercules, USA). Subsequently, the membrane blots were blocked with 5% milk or bovine serum albumin (BSA) (Sigma Aldrich; Oakville, Canada) in Tris-buffered saline with 0.1% Tween (TBS-T) for 60 min, and then washed three times with TBS-T for 30 min. The blots were then incubated overnight at 4 C with primary antibodies phospho-erk1/2 (1:1000), phospho-p38 (1:1000), phospho-akt (1:1000), and phosphor-gβ (1:5000). Gβ was included Blots were then washed thee times with TBS-T and incubated with horseradish peroxidase-labeled secondary goat anti-rabbit antisera (1:5000, Cell Signaling Technology; Danvers, USA) for 1 h at room temperature. Membranes were visualized with enhance chemiluminescence (ECL kit, GE Healthcare, UK) on the Kodak Imager 2000R. For western blot studies, phopho-protein levels were normalized to the total protein. In order to assess changes in the phosphorylation levels of these proteins, time-matched vehicle-treated controls were included, and 3-5 repeats were performed for each experiment. 2.5 Statistical analysis Data was presented as the mean ± standard error of the mean (SEM) and analyzed using 43

56 GraphPad Prism or SigmaStat Software (Systat Software, In., Chicago, USA). Statistical significance was determined using one-way or two-way ANOVA followed by a post hoc Bonferroni test. Experiments were performed as 3-5 repeats. Data was considered statistically significant when p <

57 CHAPTER 3: Sex Steroid Regulation of Estrogen Receptors and Gpr54 Gene Expression in Female Immortalized Hypothalamic Kisspeptin-expressing Cell Lines 45

58 3.1 Abstract Kisspeptin (Kiss) and its receptor, Gpr54 have emerged as fundamental gatekeepers of reproduction, acting centrally upstream of GnRH neurons in the hypothalamus. It is well established that Kiss neurons express estrogen receptors, and estradiol-mediated regulation of these neurons is nuclei-specific; where it has been demonstrated in several mammalian species that AVPV Kiss neurons are positively regulated by estradiol, while ARC neurons are inhibited. Currently, in vitro studies focusing on the regulation of genes within hypothalamic Kiss neurons is limited. To address this issue, we have generated immortalized, clonal cell lines from adultderived murine hypothalamic primary culture. We have identified two cell lines, mhypoa-50 and mhypoa-55, which exhibit endogenous Kiss-1 expression, as well as expression of Gpr54, Gpr147, and the estrogen receptors (ERα, ERβ, Gpr30). Using qrt-pcr, we report an increase in Gpr54, ERα and ERβ mrna expression at 24 h in the mhypoa-50 cell line, and at 4 h + 24 h in the mhypoa-55 cell line upon treatment with 17β-estradiol (10 nm). Studies using specific ER antagonists were completed to determine the receptor responsible for mediating changes in gene expression in the two cell lines. We found temporal involvement of estrogen receptors, as well as dependence on the specific population of kisspeptin neurons. Overall these studies elucidate the mechanisms of 17β-estradiol-mediated regulation of kisspeptin-expressing neuronal models. This information will contribute to our understanding of kisspeptin as a modulator of GnRH neurons. 3.2 Introduction Located chiefly in the hypothalamic-pituitary-gonadal (HPG) axis, is the GnRH neuronal system, acting as the central integrator and ultimate effector for reproductive functions. Adequate pulsatile GnRH secretion is necessary for the attainment and maintenance of reproductive functions, where GnRH neurons dictate the activity of reproductive systems that cycle throughout the adult reproductive life [2-4]. Although knowledge on the GnRH neuronal system has grown 46

59 substantially over the past few decades, the stimulatory neuronal systems upstream of GnRH neurons remained rather ambiguous, until recently with the discovery of kisspeptin and its receptor, Gpr54 [14, 15]. Kisspeptin and Gpr54 have been collectively recognized as indispensable mediators in the control of reproductive development and function, ranging from neonatal sexual differentiation, to regulation of GnRH and gonadotropin secretion, to the metabolic gating of puberty and adult fertility [16, 17]. Our understanding of the neurohormonal basis for activation of the HPG axis has undergone substantial conceptual modifications over the years, with emphasis being placed either on changes in the sensitivity to the feedback effects of sex steroids or modifications in the ratio between central inhibitory and stimulatory inputs to the GnRH pulse generator. It is well established that gonadal steroids, including estrogens, androgens and progesterones contribute to the dynamic control of GnRH gonadotrophin secretion via feedback regulatory loops operating within the HPG axis. Estrogens play a prominent role in reproduction, the sexual differentiation of peripheral and central brain tissue organization [51, 52], as well as energy homeostasis, neuronal growth and differentiation, mood and cognition [53, 54]. Three important estrogen receptors are known to mediate the biological actions of estrogens, participating in aspects of cellular and reproductive physiology: the two nuclear receptors ERα, ERβ, and the recently identified membrane-bound receptor, Gpr30. Furthermore, it has been shown that estrogen is capable of activating both nuclear and membrane bound receptors, where the activated receptors facilitate the overlapping or similar biological effects [74]. Studies have demonstrated a bimodal effect of E2 on the hypothalamus, having both positive and negative feedback mechanisms on GnRH neurons [57]. Both in vivo and in vitro studies reveal the negative feedback of basal E2 levels, which inhibits GnRH synthesis and secretion throughout the majority of the female cycle [58-62]. Conversely, as E2 levels increase during the late follicular phase, a stimulatory feedback system is provoked, increasing the GnRH pulse frequency and secretion to generate the GnRH surge [63-65]. While controversy surrounds 47

60 the mechanisms by which E2 regulates GnRH neurons, co-expression of GnRH and ER in mammalian GnRH neurons in vivo has been reported [67, 68]. Furthermore, the GT1-7 neuronal model demonstrates expression of both ERα and ERβ [59], and mrna expression for these receptors was found in both prepubertal and adult GnRH neurons using single cell RT-PCR [72]. Following the identification of kisspeptin in 1996, and realizing its role in reproductive physiology in 2003, Kiss signaling has emerged as a fundamental gatekeeper of reproduction, acting centrally upstream of GnRH neurons. Kisspeptins are a family of neuropeptides encoded by the Kiss-1 gene, and exert their physiological effects following activation of the cognate G protein-coupled receptor, Gpr54. Kisspeptin peptides are highly conserved and their expression has been identified in many mammalian and non-mammalian vertebrates. Kisspeptin mrna and protein has been identified in peripheral organs, and centrally in the anterodorsal POA, as well as throughout the hypothalamus, with the two major populations located in the arcuate (ARC) nucleus and the anteroventral periventricular (AVPV) nucleus [162, 183, 184, 197]. It is known that estrogen increases sensitivity of GnRH neurons to kisspeptin stimulation [109, 238, 239], and further, it is known that kisspeptin neurons are direct targets of gonadal hormones, where Kiss-1 regulation has been demonstrated. Interestingly, the nature of this regulation has demonstrated to be dependent upon the location of the kisspeptin neuron. Most notably, Kiss-1 expression in the arcuate (ARC) nucleus of the kisspeptin neurons is inhibited by estrogen, whereas AVPV Kiss-1 expression is stimulated by estrogen [183]. This has been demonstrated in rodents, sheep and monkeys, where ovariectomy and reduction in gonadal steroids, generates an increase in ARC Kiss-1 mrna expression, reversible following estradiol treatment [6, 183, 185, 188, 193]. On the other hand, results from both male and female mice indicate that early postnatal gonadectomy produces a reduction in Kiss-1 expression measured in the AVPV by 70 90% at the time of puberty, which persists throughout adulthood [197]. While the regulatory effects of estradiol on Kiss-1 expression have been thoroughly explored in many species, potential estradiol-mediated effects on other genes expressed by hypothalamic kisspeptin neurons remain unknown. 48

61 In the present study, we investigate the effects of 17β-estradiol on gene expression in hypothalamic kisspeptin neurons. Due to the heterogeneous nature and complexity of the hypothalamus, the ability to dissect these effects is confounded and would be extremely difficult to complete using the whole hypothalamus. For this reason, our lab has generated immortalized, clonal adult-derived mouse hypothalamic cell lines. These cell lines have been characterized previously (Belsham et al 2009), and we have identified two neuronal models, the mhypoa-50 and mhypoa-55, which exhibit endogenous kisspeptin expression. Here we establish that estradiol, has transcriptional effects on expression of ERα, ERβ and Gpr54 in these models of hypothalamic kisspeptin neurons. Furthermore, we utilized selective estrogen receptor antagonists in order to determine the receptor subtype responsible for mediating the transcriptional effects in our models. These findings provide the first line of evidence to suggest that 17β-estradiol induces expression of genes for estrogen receptors (ERα and ERβ) and kisspeptin receptor (Gpr54) in kisspeptin-expressing neuronal models. Furthermore, we found that the specific estrogen receptor subtype utilized is temporally dependent, as well as dependent on the population of the kisspeptin neuron. Overall, it is evident that the kisspeptin system is modulated by 17β-estradiol in our models of hypothalamic kisspeptin neurons. It is feasible to suggest that these changes in gene expression increase the responsiveness of neurons to stimulation by kisspeptin and estrogen, largely facilitating signaling of kisspeptin neurons. 49

62 A B M Hypo mhypoa-50 mhypoa-55 NTC Kiss-1 Figure 3.1. Characterization of the gene expression profile of kisspeptin-expressing, mhypoa-50 and mhypoa-55 neurons. (A) RT-PCR results of relevant reproductive neuropeptides and receptors in the whole hypothalamus (positive control) and in the indicated neuronal cell line. (+) indicates presence of a gene, and (-) indicates the absence or weak expression of a gene. (B) Representative RT-PCR screening of Kiss-1, amplicon size is 127 bp. Total RNA was isolated in used in the One-Step RT-PCR Qiagen kit with gene-specific primers. Products were visualized on a 2% agarose gel with while hypothalamus as a positive control, and non-template control (NTC) as a negative control. 50

63 3.3 Results mhypoa-50 and mhypoa-55 neurons express Kiss1 and Gpr54, as well as ERs and other reproductive peptides and receptors We have previously reported the generation of an array of clonal hypothalamic neuronal cell lines, which exhibit unique expression profiles of neuropeptides and relevant receptors (Belsham et al., 2004). In order to characterize a cell model representative of a kisspeptin-expressing cell line, RT-PCR was performed to develop a more thorough gene expression profile of the neurons. Forty-six adult male and female cell lines were screened for expression of Kiss1 mrna, and diverse expression levels were detected in the cell lines. Following initial screening for Kiss-1 gene, we identified several cell lines with the strongest expression, which were chosen for further investigation. We demonstrate that mhypoa-50 and mhypoa-55 neurons show high expression of Kiss-1 mrna (Figure 3.1B), as well as expression of Gpr54, Gpr147, ERα, ERβ and Gpr30. Screening for specific hypothalamic nuclei marker was performed in order to better identify the population of kisspeptin neurons each of the two cell lines exemplify (ARC or AVPV kisspeptin neurons). It has been previously been shown that kisspeptin neurons of the ARC nucleus coexpress substance P, neurokinin B (NKB), and dynorphin (Dyn), while those kisspeptin neurons of the AVPV co-express tyrosine hydroxylase (TH). We found that substance P, NKB and Dyn were expressed by the mhypoa-55 cell line only (Figure 3.1A), suggesting it represents a line of ARC kisspeptin neurons. These markers were not expressed by the mhypoa-50 neurons, which did express TH, supporting their identity as AVPV Kiss neurons (Figure 3.1A). The expression of Kiss-1 and the estrogen receptors indicate that these cells are appropriate models for the study of steroid hormone regulatory mechanisms of hypothalamic kisspeptin neurons. 51

64 Relative ER-a mrna expression A Control Estradiol (10 nm) Time (hours) * Relative GPR54 mrna expression B ** Time (hours) Figure 3.2. Effect of Estradiol treatment on ER-α and Gpr54 transcript levels in the mhypoa-50 neurons. Cells were treated with vehicle or 10 nm 17β-estradiol. Total RNA was collected over a 24 h time course at specified time points and changes in ER-α (A) or Gpr54 (B) mrna levels were quantified using qrt-pcr. mrna levels were normalized to the internal control, histone 3a. Data are expressed as mean ±SEM (n=4-5 independent experiments). *, P<0.05, **, P<0.01 vs. vehicle control. Statistical significance was determined by two-way ANOVA with Bonferroni s post hoc test. 52

65 3.3.2 E2 regulates Gpr54 and ERs gene expression in mhypoa-50 and mhypoa-55 neuronal models Expression of all three estrogen receptors prompted investigation into the potential regulation of Kiss-1, Gpr54, and ERs by the gonadal steroid, estradiol in the two selected neuronal models. Neurons were treated with vehicle or 10 nm estradiol over a 24 h time course, followed by measurement of Gpr54, Gpr30 and ERs mrna expression. In the mhypoa-50, we report at 24 h following estradiol treatment, an induction in the expression of Gpr54 (vehicle ± vs. E ± 0.089; P<0.01) and ERα (vehicle ± vs. E ± 0.12; P<0.05) (Figure 3.2A-B), however there was no significant change in ERβ mrna expression (data not shown). In the mhypoa-55 cell line, mrna expression of all three genes is augmented at 4 and 24 h following estradiol treatment: Gpr54 [4 h (vehicle ± vs. estrogen ± 0.155; P<0.05); 24 h (vehicle ± vs. estrogen ± 0.226; P<0.01)]; ERα [4 h (vehicle ± vs. estrogen ± 0.150; P<0.05); 24 h (vehicle ± vs. estrogen ± 0.137; P<0.01)]: ERβ [4 h (vehicle ± vs. estrogen ± 0.164; P<0.05); 24h (vehicle ± vs. estrogen ± 0.212; P<0.01) (Figure 3.3A-C). These results demonstrate regulation of Gpr54, ERα, and ERβ by estradiol in mhypoa-50 and mhypoa-55 hypothalamic cell models In the mhypoa-50 cell line, induction of mrna expression is abolished with ERα and ERβ antagonists, while the induction is lost with ERβ and Gpr30 antagonists in the mhypoa- 55 neuronal models To determine which of the estrogen responsive signaling pathways (ERα, ERβ or Gpr30) is required to mediate the transcriptional changes observed in the mhypoa-50 and mhypoa-55 cell lines following estradiol treatment, several selective estrogen receptor antagonists were employed. Briefly, neurons were pretreated for 1 h with vehicle or 1 μm of one of four 53

66 Relative ER-a mrna expression A Control Estradiol (10 nm) ** * Time (hours) Relative ER-b mrna expression B Time (hours) ** Relative GPR54 mrna expression C *** * Time (hours) Figure 3.3. Effect of Estradiol treatment on ER-α, ER-β and Gpr54 transcript levels in the mhypoa-55 neurons. Cells were treated with vehicle or 10 nm 17β-estradiol. Total RNA was collected over a 24 h time course at specified time points and changes in ER-α (A), ER-β (B), or Gpr54 (C) mrna levels were quantified using qrt-pcr. mrna levels were normalized to the internal control, histone 3a. Data are expressed as mean ±SEM (n=4-5 independent experiments). *, P<0.05, **, P<0.01 vs. vehicle control. Statistical significance was determined by two-way ANOVA with Bonferroni s post hoc test. 54

67 antagonists: ERα antagonist (MPP dihydrochloride), ERβ antagonist (PHTPP), ER antagonist (ICI ), Gpr30 antagonist (G-15). Neurons were then treated with either vehicle or estradiol (10 nm). Additionally, other plates of the same neurons were treated an ERα-selective agonist (PPT), or ERβ-selective agonist (DPN), serving as positive controls. RNA was harvested at 4 and 24 h following estradiol treatment the times corresponding to the significant induction in gene expression previously observed. In the mhypoa-50 cell line, we report that at 24 hours following estrogen treatment, ERα and ERβ both facilitate the upregulation of Gpr54, ERα and ERβ mrna expression. This is demonstrated by results which reveal that only when both ERα and ERβ receptors are antagonized, is the induction in gene expression lost (Figure 3.4A-B). Gene induction of ERα ensued with the use of MPP (vehicle ± vs. estradiol ± ; P<0.001), PHTPP (vehicle ± vs. estradiol 1.20 ± 0.099; P<0.001), and G-15 (vehicle ± vs. estradiol ± 0.103; P<0.001) (Figure 3.4A). However, when neurons were pre-treated with ICI , antagonizing both ERα and ERβ, there was no change in gene expression subsequent to estradiol treatment, signifying the necessity for ERα and ERβ to be functional. Similar results were found for expression of Gpr54. In the mhypoa-55 cell line, results demonstrate that at 4 hours Gpr30 and ERβ are required to mediate the upregulation of Gpr54, ERα and ERβ mrna expression. However, at 24 hours, ERβ, without Gpr30, is responsible for mediating these transcriptional changes (Figure 3.5A-C). Expression of ERα was induced with all pre-treatments and subsequent estradiol treatments, with the exception of ERβ antagonism [4 hours: MPP (vehicle ± vs. estradiol ± ; P<0.01); ICI (vehicle ± vs. estradiol ± 0.148; P<0.05); and 24 hours: MPP (vehicle ± vs. estradiol ± 0.197; P<0.001); G-15 (vehicle ± vs. estradiol ± 0.213; P<0.05)]. (Figure 3.5A). Similar results were measured with expression of Gpr54 and ERβ. These findings suggest that estradiol increases Gpr54, ERα and ERβ gene expression, via activation of different estrogen-dependent signaling pathways, dependent on the specific cell line or population of kisspeptin neurons being studied. Furthermore, as is evident from observations in 55

68 Relative ER-a mrna expression A DMSO EthOH Estradiol (10 nm) MPP Dihydrochloride PHTPP G-15 ICI PPT DPN *** *** *** *** ** *** Relative GPR54 mrna expression B DMSO EthOH Estradiol (10 nm) MPP Dihydrochloride PHTPP G-15 ICI PPT DPN *** ** *** *** ** * *** Figure 3.4. Effects of estrogen receptor antagonists on estradiol-mediated induction of ER-α and Gpr54 mrna expression in mhypoa-50 neurons. Cells were pre-treated for 1 h in the presence of one specific estrogen receptor antagonist (1 um) or vehicle control, followed by treatment with estradiol (10 nm), or specific estrogen receptor agonist (10 nm), or vehicle over a 24 h time course. ER-α and Gpr54 mrna expression was determined using real-time RT-PCR and levels were normalized to the internal control, histone 3a. Results shown are expressed as mean ± SEM (n=3-5 independent experiments). *, P<0.05, **, P<0.01, ***, P<0.001 vs. vehicle control. Statistical analysis was calculated by two-way ANOVA. White bars, control vehicle (H20, DMSO in the presence or absence of estrogen receptor antagonists); black bars, treatment (estradiol in the presence or absence of antagonists). 56

69 the mhypoa-50 cell line, while the nuclear estrogen receptor is required to induce gene expression over a longer period of time, the membrane-bound receptor mediates the earlier and more rapid effects on gene transcription. 3.4 Discussion Several years following its initial discovery as a tumor suppressor gene, kisspeptin has gained recognition as an important regulator of the HPG axis and reproductive development through its potent stimulation of GnRH secretion [6, 30, 162, 168]. In vivo animal studies have been essential in developing our current knowledge of Kiss-1 regulation and kisspeptin secretion by central and peripheral factors during pre-pubertal, pubertal and adult stages of development. Despite knowledge of kisspeptin and its significant role in reproduction however, there remains a scarcity of studies that focus on gene expression by regulators of hypothalamic kisspeptinexpressing neurons, which can be attributed to the previous unavailability of a suitable model for use in in vitro studies. For this reason, we sought to establish kisspeptin-expressing cell models from the adult-derived mouse hypothalamus, to be utilized for the study of kisspeptin neuronal regulation. The results obtained from our studies provide better understanding of the regulation of gene expression in kisspeptin neurons in response to fluctuating gonadal hormone levels. Overall, a variety of different molecular techniques were utilized for the purpose of characterizing two different kisspeptin-expressing neuronal cell models, the mhypoa-50 and the mhypoa-55. In the present study, an array of immortalized, clonal, hypothalamic cell lines from postpubertal female adult mouse hypothalamus were used to establish kisspeptin-expressing cell models. The cell lines were screened for expression of Kiss-1 mrna, and RT-PCR was used to identify strong expression in the mhypoa-50 and the mhypoa-55 cell lines (Figure 3.1B), which were also found to express mrna of genes that are relevant to reproductive signaling pathways, such as the kisspeptin and estrogen receptors. Important to note, screening of our 57

70 Relative ER-a mrna expression A hr 24 hr ** NS ** ** ** NS *** *** *** *** * * Relative ERb mrna expression B NS *** *** 4 hr 24 hr ** *** * NS *** *** *** *** *** ** Relative GPR54 mrna expression C DMSO EthOH 17-b Estradiol MPP Dihydrochloride PHTPP G-15 ICI PPT DPN NS ** *** 4 hr 24 hr NS *** * ** *** *** *** *** *** Figure 3.5. Effects of estrogen receptor antagonists on estradiol-mediated induction of ERα, ER-β and Gpr54 mrna expression in mhypoa-55 neurons at 4 h and 24 h. Cells were pre-treated for 1 h in the presence of one specific estrogen receptor antagonist (1 um) or vehicle control, followed by treatment with estradiol (10 nm), or specific estrogen receptor agonist (10 nm), or vehicle over a 24 h time course. RNA was collected at 4 h and 24 h, and ER-α (A), ER-β (B) and Gpr54 (C) mrna expression was determined using real-time RT-PCR and levels were normalized to the internal control, histone 3a. Results shown are expressed as mean ± SEM (n=3-5 independent experiments). *, P<0.05, **, P<0.01, ***, P<0.001 vs. vehicle control. Statistical analysis was calculated by two-way ANOVA. White bars, control vehicle (H2O, DMSO in the presence or absence of estrogen receptor antagonists); black bars, treatment (estradiol in the presence or absence of antagonists). 58

71 hypothalamic cell lines from mouse embryonic-derived hypothalami had extremely low levels of kisspeptin mrna (data not shown), and this expression analysis complies with in vivo reports which reveal pre-pubertal kisspeptin expression to be far lesser in comparison to pubertal and adult levels [30, 109], revealing the importance of gonadal steroids on Kiss-1 expression in the hypothalamus. High levels of kisspeptin mrna and protein have been identified in rodent hypothalamic nuclei including the ARC, AVPV and periventricular nucleus (PVN), with lower expression levels in found in the anterodorsal POA [162, 183, 184, 197]. Two major populations of kisspeptin neurons, in the AVPV and the ARC have been thoroughly explored [162, 183, 184], and found to differ in their co-expression profiles with other neuropeptides and neurotransmitters. The most consistent co-localization for kisspeptin neurons, is that seen in the ARC nucleus, where the majority of kisspeptin neurons also express dynorphin (Dyn) and neurokinin B (NKB), thus leading to the acronym KNDy neurons [192]. Additionally, ARC kisspeptin neurons have been found to co-express substance P [214], a peptide belonging to the tachykinin peptide family. On the other hand, kisspeptin neurons in the AVPV of mice have demonstrated co-expression with met-enkephalin [284, 285], an endogenous opioid peptide, and TH [205, 285], the ratelimiting enzyme necessary for dopamine synthesis. Screening revealed expression of dynoprhin, NKB and substance P in the mhypoa-55 cell line, suggesting that the mhypoa-55 cell line represents ARC nucleus kisspeptin neurons. The lack of NKB, Dyn and substance P expression, and expression of met-enkephalin in the mhypoa-50 cell line, indicates that it is not representative of a population of ARC nucleus kisspeptin neurons, rather, it may embody AVPV neurons, or another population of hypothalamic kisspeptin neurons. Further screening revealed expression of ERα and ERβ, which coincides with findings that reveal nearly all ARC and AVPV (98-99%) hypothalamic kisspeptin neurons express ERα mrna, and 25-30% express ERβ mrna [183] (Figure 3.1A). Overall screening has revealed that the mhypoa-50 and mhypoa- 59

72 55 neuronal models characterize two distinct populations of hypothalamic kisspeptin neurons. Following detection of estrogen receptor mrna in the two neuronal models, we were prompted to investigate the potential responsiveness of our cell lines to estrogen. We measured an increase in c-fos mrna expression with 17β-estradiol treatment, establishing that both cell lines are sensitive to estradiol stimulation (data not shown). We therefore explored the 17β-estradiolmediated regulation of genes for the estrogen and kisspeptin receptors expressed by our cell lines. The increase in ERα, ERβ and Gpr54 mrna expression measured in both cell lines suggest that estrogen has stimulatory effects on these genes, regardless of the neuronal population involved (Figure 3.2. and Figure 3.3.). Our results are congruent with findings from a recent study by Jacobi and colleagues who used GT1-7 cells to demonstrate induction of Gpr54 gene expression (6-fold) at 24 h following estradiol treatment [286]. Therefore, it appears as though regulation of Gpr54 by estradiol in kisspeptin-expressing neurons is similar to regulation in GnRH neurons, thus signifying the role of estrogens to prime hypothalamic neurons for responsiveness to kisspeptin stimulation. Additionally, increased expression of the nuclear estrogen receptors indicates that neurons may become more sensitive to future stimulation by estrogen, revealing a positive feedback method by which estrogen facilitates neuronal responses to itself. Furthermore, high levels of circulating estrogens are known to exist at the onset of puberty, and during the late follicular phase of the menstrual cycle. If increased estrogen coincides with more sensitive kisspeptin neurons, it is likely that during times of physiological development when estrogen is high, kisspeptin neurons are most active and signal more efficiently to GnRH neurons downstream. We therefore suggest that estrogen contributes greatly in the maintenance of reproductive function at puberty and throughout adulthood. Our studies with selective estrogen receptor antagonists reveal that estrogen mediates effects of gene induction through different mechanisms which are temporally-dependent, and contingent on the neuronal population. In the mhypoa-50 cell line, estrogen requires one of ERα 60

73 or ERβ to induce gene expression, where one receptor is sufficient to mediate the full transcriptional effects of estrogen. This is evident from results that reveal during antagonism of one nuclear receptor, significant up-regulation of mrna expression is maintained as the other nuclear receptor is capable of signaling the effects of estrogen. [30, 49, 57, 81, 112, 132, 138, 193]. However if both receptors are blocked, estrogen has no effects on gene transcription of ERα or Gpr54 (Figure 3.4). This suggests a shared role of the nuclear estrogen receptors, where they synergistically mediate the same biological function. In the mhypoa-55 cell line, at 4 h Gpr30 and ERβ are involved in the up-regulation of mrna expression, however, at 24 h ERβ acts alone (Figure 3.5). These results expose the significant role of the membrane-bound receptor, Gpr30 in mediating the more transient effects of estrogen in the hypothalamus, which act through rapid protein signaling cascades, leading to phosphorylation of transcription factors and co-activator molecules or the nuclear receptors themselves to mediate gene transcription. This finding is in agreement with previous reports suggesting the importance of Gpr30 in the negative feedback mechanism of estrogen on GnRH pulsatility [1], which is facilitated by ARC kisspeptin neurons. The present study suggests that the mhypoa-50 and mhypoa-55 cell lines are representative of two different functional populations of kisspeptin-expressing hypothalamic neurons, where the mhypoa-55 neuronal model may represent ARC kisspeptin neurons based on expression of NKB, Dyn and Substance P. Furthermore, our work demonstrates that estrogen upregulates expression of ERα, ERβ and Gpr54, all of which play a pivotal role regulating fertility. We report involvement of several different estrogen receptor subtypes to mediate changes in gene expression induced by 17β-estradiol. In addition, the estrogen receptor activated by 17β-estradiol, involved in regulating gene expression is temporally based and determined by the population of the kisspeptin neurons. We established that either ERα or ERβ were necessary in the population represented by mhypoa-50 cells, whereas Gpr30 and ERβ were required by the mhypoa-55 kisspeptin neuronal model. We have demonstrated the effect of 17β-estradiol on hypothalamic 61

74 kisspeptin-expressing neuronal models, and overall the results suggest that the physiologic effects of estradiol on the function of the reproductive axis is mediated in part by the modulation of Gpr54, ERα and ERβ expression in kisspeptin-expressing neurons. Because it is known that Kiss- 1 expression is modulated by estrogen, our findings here may reveal a additional mechanism by which estrogen regulates the HPG axis via alterations in the sensitivity of kisspeptin neurons to stimuli, subsequently altering their output; a mechanism necessary to maintain GnRH pulsatility and reproductive function. 62

75 CHAPTER 4: Hypothalamic Neuropeptide Kisspeptin and GnIH Regulate Gene Expression and MAPK Signaling Pathways in Immortalized Hypothalamic Kisspeptin-expressing Cell Lines 63

76 4.1 Abstract Fertility is a complex and highly regulated process dependent on the orchestration of hypothalamic neuropeptides and peripheral hormones. Signals converge on gonadotropinreleasing hormone (GnRH) neurons, positioned at the pinnacle of the hypothalamic-pituitarygonadal (HPG) axis. Kisspeptin (Kiss) and its receptor, Gpr54, have emerged as fundamental gatekeepers of reproduction, acting centrally upstream of GnRH neurons. It is well established that Kiss neurons express estrogen receptors, and estradiol-mediated regulation of these neurons is nuclei-specific. Further, subpopulations of Kiss neurons have been found to express Gpr54 and the gonadotropin-inhibitory hormone (GnIH) receptor, Gpr147, suggesting additional mechanisms of auto-regulation and inhibition in Kiss neurons. Currently, in vitro studies focusing on the regulation of genes expressed within hypothalamic Kiss neurons are limited. To address this issue, we have generated immortalized, clonal cell lines from adult-derived murine hypothalamic primary culture. We have identified two cell lines, mhypoa-50 and mhypoa-55, which exhibit endogenous Kiss expression, as well as expression of Gpr54 and Gpr147. Using qpcr, we report an induction of Gpr54 and Kiss-1 mrna expression following Kiss-10 treatment in both cell lines. Western blot analyses were performed to delineate the Kiss-10- mediated mechanisms controlling transcription of the genes studied, and we report induction of several MAP Kinase pathways, including ERK1/2 and p38 pathway. Following GnIH treatment, in the mhypoa-50 neurons, we report a significant decrease in Kiss-1 and Gpr54 mrna expression, whereas in the mhypoa-55 neurons, there was suppression of Kiss-1 expression only. This is the first study to report auto-regulation of kisspeptin neurons, and further, to expose mechanisms of GnIH-mediated kisspeptin neuronal inhibition. Furthermore, these studies will expand our knowledge of the regulation of Kiss neurons, which will be essential for better understanding of kisspeptin as a modulator of GnRH neurons and the HPG axis. 64

77 4.2 Introduction Although GnRH and its central role in reproduction were exposed over 40 years ago [287], further studies are required to completely understand the afferent neuronal populations and pathways through which hormonal and environmental signals regulate the secretion of GnRH. Since the realization of the central role of kisspeptin signaling in reproduction [10, 180, 181], studies have focused on hypothalamic kisspeptin as an afferent regulator of GnRH secretion. Several convincing studies have reported the ability of kisspeptin to convey feedback effects of steroidal hormones on secretion of GnRH during puberty [166, 288], throughout the estrous cycle [289], and during seasonal reproductive transitions [290]. More recently, another neuropeptide, GnIH was discovered in 2000, and identified as a potent inhibitor of gonadotropin secretion acting at the level of the hypothalamus and the pituitary [150, 291]. The complex network of neuroendocrine circuitries, and combination of stimulatory and inhibitory inputs, is essential for the correct timing of puberty onset and overall functioning of the HPG axis, however, due to the interconnectivity and overlapping of partially redundant pathways, pathophysiologies can arise from perturbations at any level, presenting as a concern in reproductive maturation. Following its identification in 1996 [14], and realizing its role in reproductive physiology in 2003 [10, 180, 181], kisspeptin has become recognized as the most potent upstream stimulator of GnRH neurons in the hypothalamus and therefore has emerged as a fundamental gatekeeper of reproduction [109, 162, 168, 199, 225, 233, 288]. Kisspeptins are a family of neuropeptides encoded by the Kiss-1 gene, belonging to the family of RF-amide peptides [16, 17, 177]. Kisspeptin peptides are highly conserved and their expression has been identified in many mammalian and non-mammalian vertebrates. Kisspeptin mrna and protein have been identified in peripheral organs, and centrally in the anterodorsal POA, as well as several different hypothalamic nuclei, with the two major populations of kiss neurons being situated in the arcuate (ARC) nucleus and the anteroventral periventricular (AVPV) nucleus [162, 183, 184, 197]. 65

78 Additionally, Gpr54 mrna and protein expression have been identified in many central brain regions and peripheral organs, with highest expression in the hypothalamus and amygdala [15]. More specifically within the hypothalamus, Gpr54 mrna expression has been localized to the ARC, dorsomedial hypothalamic nucleus and the lateral hypothalamic area [15]. Despite the identification of Gpr54 expression in kisspeptin neurons, the effects of kisspeptin stimulation on Kiss neurons has yet to be explored. Following the discovery of Kiss-1, another novel RFamide peptide, GnIH was isolated and characterized from the Japanese quail, having the unique ability to inhibit gonadotropin release from the pituitary gland [150]. Immunohistochemical studies localized the peptide to cell bodies in the PVN and terminals in the ME [150]. In order to elucidate the mode of action of GnIH, the receptor was identified and its expression and binding activity was characterized in the quail brain. GnIH receptor is a novel seven transmembrane G-protein coupled receptor, Gpr147, and binds GnIH peptide in a concentration-dependent manner [153]. Gpr147 expression has been demonstrated in regions of the hypothalamus, pituitary and gonads [156, 292]. In addition to its known actions on the pituitary, it was shown in sparrows that in vivo GnIH treatment rapidly inhibits GnRH-induced LH release [154], and it is therefore reasonable to suggest that GnIH acts via the GnIH receptor at the level of the hypothalamus to inhibit GnRH release. The mammalian homologue to the avian GnIH, has been identified as RFRP-3, and also found to be an inhibitory regulator of hypothalamic GnRH neurons in mammals, preventing gonadotropin secretion and silencing of the HPG axis [155, 156]. Kisspeptin and GnIH neurons in the rodent are known to appose GnRH neurons in the hypothalamus where Gpr54 and Gpr147 are expressed, providing excitatory and inhibitory inputs respectively [109, 156, 163, 262, 291]. Due to the paucity of current knowledge on the regulation of GnIH and Gpr147 in the central nervous system, a recent study aimed to quantify the levels of expression of the two genes throughout the brain, and it was reported that Gpr147 expression was highest in the AVPV nucleus compared to all other brain regions [158]. From this finding, it was 66

79 suggested that in addition to its direct actions on GnRH neurons, GnIH may act on kisspeptin neurons where Gpr147 is expressed, to indirectly regulate GnRH neurons. In the present study, we sought to investigate the effects of Kisspeptin (Kiss-10) and GnIH on gene transcription in hypothalamic kisspeptin neurons, in order to identify novel mechanisms of regulation in kisspeptin neurons. We believe that these mechanisms of regulation will further modulate the sensitivity and enhance signaling of the kisspeptin network. Due to the heterogeneous nature and complexity of the hypothalamus, the ability to dissect these effects is confounded and would be extremely difficult. For this reason, our lab has generated immortalized, clonal adult mouse hypothalamic cell lines. These cell lines have been characterized previously, and we have identified two cell lines, mhypoa-50 and mhypoa-55 cell lines, which exhibit endogenous kisspeptin expression, as well as expression of Gpr54 and Gpr147. Here we establish that Kiss-10 and GnIH do have transcriptional effects on expression of Kiss-1 and Gpr54 in hypothalamic kisspeptin neurons. With the use of western blot analysis, we have been able to elucidate the signaling cascade activated by Kiss-10 treatment in our cell lines. Taken together, these findings provide the first line of evidence to suggest that auto-regulatory mechanisms exist within kisspeptin neurons, whereby secreted kisspeptin can activate receptors on the same neuron from which it was released, inducing gene expression and acting in a feedforward manner. Additionally, we suggest that GnIH may be a regulator of GnRH neurons indirectly, acting via an intermediate population of kisspeptin neurons. Overall, the kisspeptin system appears to be modulated by hypothalamic neuropeptides, which alter gene expression in order to enhance or attenuate kisspeptin signaling, and overall facilitate reproductive function. 4.3 Results mhypoa-50 and mhypoa-55 neurons express Kiss1, Gpr54, and Gpr147 as well as other reproductive peptides and receptors 67

80 We have previously reported the generation of an array of clonal hypothalamic neuronal cell lines, which exhibit unique expression profiles of neuropeptides and relevant receptors [276, 277]. We have previously characterized two cell models representative of kisspeptin-expressing cell lines, through the use of RT-PCR and development of a comprehensive gene expression profile of the neurons. Briefly, we screened forty-six adult male and female cell lines for expression of Kiss-1 mrna, and from those cell lines, we were able to obtain two adult female mouse cell lines which demonstrate the strongest expression of Kiss-1. We demonstrate that mhypoa-50 and mhypoa-55 neurons show high expression of Kiss-1 mrna, as well as Gpr54, Gpr147. The expression of Kiss-1 and its receptor, as well as the GnIH receptor indicate that these cell lines are appropriate models for use to study of regulation of hypothalamic kisspeptin neurons by Kiss-10 and GnIH treatments. Furthermore, expression of these receptors suggests that an auto-regulatory mechanism, and an inhibitory input may exist within the hypothalamic kisspeptin neuronal pathway a question which currently remains unexplored Kiss-10 peptide treatment regulates Kiss1 and Gpr54 mrna expression in mhypoa-50 and mhypoa-55 cell lines Due to expression of Gpr54 in the mhypoa-50 and mhypoa-55 kisspeptin neurons, we investigated the potential for a kisspeptin auto-regulatory mechanism. Cells were treated with kiss-10 peptide (10nM) over a 24 h time course and quantitative RT-PCR was performed to measure gene expression. In the mhypoa-50 cell line, kiss-10 treatment induces Kiss-1 mrna expression at 24 h (vehicle ± vs. estrogen ± 0.206; P<0.01), although Gpr54 mrna expression was not altered (Figure 4.1A-B). Conversely, in the mhypoa-55 cell line, kiss-10 treatment augmented mrna expression at 24 h of Kiss-1 (vehicle ± vs. Kiss ± 0.155; P<0.001) and Gpr54 (vehicle ± vs. Kiss ± 0.058; 68

81 A Relative Kiss1 mrna expression Control Kiss-10 (10 nm) ** ** Time (hours) B Relative GPR54 mrna expression Time (hours) Figure 4.1. Effect of Kiss-10 treatment on Kiss-1 and Gpr54 transcript levels in the mhypoa-50 neurons. Cells were treated with vehicle or 10 nm Kiss-10. Total RNA was collected over a 24 h time course at specified time points and changes in Kiss-1 (A) or Gpr54 (B) mrna levels were quantified using qrt-pcr. mrna levels were normalized to the internal control, histone 3a. Data are expressed as mean ±SEM (n=4-5 independent experiments). *, P<0.05, **, P<0.01 vs. vehicle control. Statistical significance was determined by two-way ANOVA with Bonferroni s post hoc test. 69

82 A Control Kiss10 (10nM) Relative Kiss1 mrna expression Time (hours) ** Relative GPR54 mrna expression B Time (hours) ** Figure 4.2. Effect of Kiss-10 treatment on Kiss-1 and Gpr54 transcript levels in the mhypoa-55 neurons. Cells were treated with vehicle or 10 nm Kiss-10. Total RNA was collected over a 24 h time course at specified time points and changes in Kiss-1 (A) or Gpr54 (B) mrna levels were quantified using qrt-pcr. mrna levels were normalized to the internal control, histone 3a. Data are expressed as mean ±SEM (n=4-5 independent experiments). *, P<0.05, **, P<0.01 vs. vehicle control. Statistical significance was determined by two-way ANOVA with Bonferroni s post hoc test. 70

83 P<0.001) (Figure 4.2A-B). These findings suggest that kiss-10 acts on kisspeptin neurons where Gpr54 is expressed to further induce Kiss-1 expression, as well as Gpr54 expression Kiss-10 treatment activates MAPK intracellular signaling cascades in mhypoa-50 and mhypoa-55 cell lines In order to determine the signaling pathways activated upon treatment with kisspeptin in our endogenous Gpr54-expressing hypothalamic cell lines, neurons were treated with kiss-10 (10 nm) over a 1 h time course. We used western blot analysis to investigate the potential activation of different MAP kinase pathways by kiss-10 treatment. In the mhypoa-50 cell line, we found significant phosphorylation of ERK1/2 protein at 60 min (vehicle ± vs. Kiss ± 0.204; P<0.01) (Figure 4.3A). In the mhypoa-55 cell line, there was also a significant increase in ERK1/2 phosphorylation at 5 min (vehicle ± vs. Kiss ± 0.162; P<0.01) and 60 min (vehicle ± vs. Kiss ± 0.172; P<0.001) (Figure 4.3A). Following Kiss-10 treatment, there was however no change in Akt protein phosphorylation in either mhypoa-50 or mhypoa-55 cell lines (Figure 4.3B). In addition, we report an up-regulation of p38 phosphorylation at 5 min, followed by a down-regulation at 15 min in mhypoa-55 cells (Figure 4.4), although no change was measured for the mhypoa-50 cell line. Together these results obtained in mhypoa-55 cell line indicate that upon binding to Gpr54, kisspeptin activates the p38 and ERK1/2 protein signaling cascades. On the other hand, the ERK1/2 pathway alone is activated in the mhypoa-50 cell line, with no activation of p38 pathway GnIH peptide treatment regulates Kiss-1 and Gpr54 mrna expression in mhypoa-50 and mhypoa-55 cell lines Following RT-PCR screening of our two cell lines, mhypoa-50 and mhypoa-55, we report that 71

84 Control A Control mhypoa-50 * 2.0 * * 2.0 ** Relative pakt expression Relative pakt expression Time (min) Time (min) B Kiss10 (10nM) mhypoa-55 Relative perk expression Relative perk expression A Kiss10 (10nM) Time (min) Time (min) Figure 4.3. Kisspeptin phosphorylates ERK1/2 protein but not Akt protein in mhypoa-50 and mhypoa-55 cell lines. mhypoa-50 and mhypoa-55 neurons were serum starved for 4 h prior to treatment with kisspeptin (10 nm) over a 1 h time course. Cell lysates were harvested at indicated time points. Western blot analysis was performed and imaged with enhanced chemiluminescence using phospho-specific antibodies directed against ERK1/2 (A) and Akt (B). Protein phosphorylation levels are relative to total protein, and expressed as mean ±SEM (n=3-5 independent experiments). *, P<0.05, **, P<0.01 vs. vehicle control. Statistical significance was determined by two-way ANOVA with Bonferroni s post hoc test. 72

85 Control Kiss-10 Relative p-p38 expression *** *** Time (min) Figure 4.4. Kisspeptin modifies phosphorylation status of p38 protein in mhypoa-55 cell lines. mhypoa-55 neurons were serum starved for 4 h prior to treatment with kisspeptin (10 nm) over a 1 h time course. Cell lysates were harvested at indicated time points. Western blot analysis was performed and imaged with enhanced chemiluminescence using phospho-specific antibodies directed against ERK1/2 (A) and Akt (B). Protein phosphorylation levels are relative to total protein, and expressed as mean ±SEM (n=3-5 independent experiments). ***, P<0.001 vs. vehicle control. Statistical significance was determined by two-way ANOVA with Bonferroni s post hoc test. 73

86 in addition to Gpr54, both cell lines express Gpr147. As a result, cells were treated with GnIH peptide (100 nm) over a 24 h time course and RNA was isolated at the indicated time points, followed by quantitative RT-PCR for measurement of mrna expression. In the mhypoa-50 cell line, results reveal a suppression of Kiss-1 mrna [4 h (vehicle ± vs. GnIH ± ; P<0.05), 8 h (vehicle ± vs. GnIH ± ; P<0.001), and 24 h (vehicle ± vs. GnIH ± ; P<0.01)], as well as of Gpr54 mrna at 24 h (vehicle ± vs. GnIH ± 0.053; P<0.05) (Figure 4.5A-B). Furthermore, the trend of decreasing gene expression following GnIH treatment (compared to vehicle treatment) is evident as early as 1 h. In the mhypoa-55 cell line, a down-regulation was measured for expression of Kiss-1 mrna [4 h (vehicle ± vs. GnIH ± ) and 24 h (vehicle ± vs. GnIH ± 0.055)], although we report no change in the expression of Gpr54 (Figure 4.6A-B). These results clearly establish the responsiveness of both of our kisspeptin neuronal models to GnIH treatment, demonstrating transient attenuation of Kiss-1 and Gpr54 gene expression. 4.4 Discussion Kisspeptin is widely recognized as a principal hypothalamic neuropeptide in the context of reproductive physiology, due to its ability to potently activate GnRH neurons directly, in turn stimulating the HPG axis [109, 162, 165, 168, 225, 293]. The stimulatory effects of kisspeptin on GnRH biosynthesis and secretion have been thoroughly explored in animal studies. More interesting however, is that Gpr54 expression has been revealed in several hypothalamic neuronal populations besides GnRH neurons, including expression by Kiss neurons themselves. For this reason, we predict that kisspeptin neurons are regulated in part, by auto-regulatory signaling mechanisms, an area which has yet to be investigated. Our studies demonstrate that mhypoa-50 and mhypoa-55 kisspeptin-expressing 74

87 A Control GnIH (100 nm) Relative Kiss1 mrna expression 1.25 * 1.00 *** ** Time (hours) Relative GPR54 mrna expression B Time (hours) * Figure 4.5. Effect of GnIH treatment on Kiss-1 and Gpr54 transcript levels in the mhypoa- 50 neurons. Cells were treated with vehicle or 10 nm Kiss-10. Total RNA was collected over a 24 h time course at specified time points and changes in Kiss-1 (A) or Gpr54 (B) mrna levels were quantified using qrt-pcr. mrna levels were normalized to the internal control, histone 3a. Data are expressed as mean ±SEM (n=4-5 independent experiments). *, P<0.05, **, P<0.01 vs. vehicle control. Statistical significance was determined by two-way ANOVA with Bonferroni s post hoc test. 75

88 neuronal models are stimulated by kisspeptin treatment (Figure 4.1 and Figure 4.2), to activate potential feed-forward mechanisms, and increase expression of Kiss-1 and Gpr54 genes. These findings suggest that auto-regulation may be a facet of kisspeptin neuronal regulation. Kisspeptin peptide can bind to its receptor expressed by the same neurons from which the peptide was secreted, in order to activate the receptor signaling pathway, and triggering an increase in Kiss-1 gene expression. While Kisspeptin-Gpr54 signaling has been demonstrated in GnRH GT1-7 neurons and shown to increase GnRH mrna expression [221], this pathway has not been studied in kisspeptin neurons. In addition to the upregulation of Kiss-1 gene expression, this autoregulatory mechanism caused increased Gpr54 gene expression in kisspeptin-expressing neuronal models. In physiological context, an increase in Gpr54 expression may be necessary to facilitate sustained responsiveness of kisspeptin neurons to kisspeptin stimulation, enabling continued peptide release upon stimulation. As secreted and circulating kisspeptin levels rise, Gpr54 at the membrane must retain ability to transduce ligand signaling efficiently. A study in 2009 performed in human embryonic kidney (HEK) 293 cells, revealed Gpr54 to be a constitutively active receptor [294]. The study reported basal Gpr54 activity to be at about 5% of the maximal kisspeptin-induced activity, with its activity under molecular regulation by G protein-coupled receptor Kinase-2 (GRK2) and β-arrestin [294]. This is not completely surprising, as constitutive activity has been described for over 60 wild-type GPCRs [295]. In addition, Gpr54 activity is thought to be regulated by GRK2 and β-arrestin, at the level of receptor desensitization, internalization, and degradation [294]. While very little research has been completed studying this phenomenon at the molecular level, it has been demonstrated that upon activation, Gpr54 does undergo agonist-dependent desensitization, where the receptor is internalized via a β-arrestin /clathrin-dependent mechanism [294]. The physiological significance of this rapid receptor desensitization and internalization remain unknown, however, it may justify increased Gpr54 mrna expression following Kiss-10 stimulation in the present study. Here we suggest that following ligand binding, Gpr54 rapidly leaves the membrane, becoming 76

89 A Control GnIH (100nM) Relative Kiss1 mrna expression ** * Time (hours) Relative GPR54 mrna expression B Time (hours) Figure 4.6. Effect of GnIH treatment on Kiss-1 and Gpr54 transcript levels in the mhypoa- 55 neurons. Cells were treated with vehicle or 10 nm Kiss-10. Total RNA was collected over a 24 h time course at specified time points and changes in Kiss-1 (A) or Gpr54 (B) mrna levels were quantified using qrt-pcr. mrna levels were normalized to the internal control, histone 3a. Data are expressed as mean ±SEM (n=4-5 independent experiments). *, P<0.05, **, P<0.01 vs. vehicle control. Statistical significance was determined by two-way ANOVA with Bonferroni s post hoc test. 77

90 desensitized. As a compensatory mechanism to overcome lost receptors at the membrane, stimulated neurons initiate Gpr54 gene transcription (and eventual translation to protein), allowing kisspeptin-expressing neurons to remain sensitive and be maximally activated upon subsequent kisspeptin stimulation. It is well-documented that kisspeptin binds to the Gpr54, activating the G q/11 protein intracellularly. Activation of this G protein precedes activation of PLC, causing hydrolysis of PIP2, inositol-(1,4,5)-triphosphate (IP3)-mediated intracellular Ca 2+ release, and activation of protein kinase C (PKC) by diacyglycerol (DAG) [16, 17, 177]. Furthermore, receptor activation was found to be associated with arachidonic acid release, and activation of various MAPK pathways [16, 17, 177, 200, 238, 296, 297]. Studies using cell models derived from different tissues have demonstrated that the specific pattern of MAPK pathways activated following kisspeptin-induced activation of Gpr54, is highly dependent on the cellular context [ ]. All of the initial studies that characterized the nature of Kisspeptin-Gpr54 signaling however, used heterologous cell models expressing the rat or human Gpr54 [16, 17, 177], or were performed in GnRH neuronal models such at the GN11 and GT1-7 cell lines [238], however, this signaling has yet to be investigated in hypothalamic kisspeptin-expressing neuronal models. Our western blot analysis results indicate that kisspeptin binds to Gpr54 in order to activate a unique range of signaling pathways in our immortalized hypothalamic kisspeptin expressing cell models. We found significant phosphorylation of ERK1/2 in the mhypoa-50 and mhypoa-55 cell lines, at 15 and 60 min, and 5 and 60 min respectively (Figure 4.3A), findings which are supported by previous work [16]. In addition, kiss-10 treatment demonstrates activation of the p38 pathway in mhypoa-55 neuronal model only (Figure 4.4), without involvement of the Akt pathway in either cell line (Figure 4.3B); findings that are in agreement with previously published data using heterologous cell lines [16, 17, 177]. 78

91 Following screening of our two kisspeptin-expressing cell lines, we demonstrate expression of Gpr147 by populations of kisspeptin neurons in vitro. This finding is supported by immunoreactive studies, which demonstrate that in proestrous female rats, 19% of the AVPV kisspeptin neurons analyzed exhibit close contact with GnIH fibers [265]. Furthermore, while 33% of GnRH neurons analyzed expressed Gpr147, 16% of AVPV kisspeptin neurons were found to express Gpr147 in diestrous females [265]. This further suggests a role of GnIH in providing negative input to kisspeptin neurons, providing an indirect pathway to modulate expression of GnRH. We therefore examined if GnIH has any role in regulating hypothalamic kisspeptin-expressing neurons. We observed suppression of Kiss-1 or both Kiss-1 and Gpr54 mrna expression in our mhypoa-50 and mhypoa-55 cell lines following treatment with 100 nm GnIH a trend that is apparent as early as 1 h following treatment (Figure 4.5 and Figure 4.6). This suggests that GnIH not only exerts actions on GnRH neurons to attenuate GnRH synthesis and secretion, but in addition, it acts on kisspeptin neurons to decrease kisspeptin- Gpr54 signaling. In turn, we propose that this reduces GnRH neuronal activation, putting the HPG axis into a quiescent state. Our findings provide support for GnIH negatively influencing function of GnRH neurons through direct synaptic connections, and indirectly via populations of kisspeptin neurons. Overall, we have elucidated two novel mechanisms of kisspeptin-neuronal regulation including autoregulation and neuropeptide-induced inhibition. These findings indicate that kisspeptin neurons are a highly complex system, acting as integrators and facilitators, collecting information from hypothalamic neuropeptides and generating output that is conveyed onto the GnRH neurons, and thus enabling the fine-tuning to precisely coordinate the timing of neuroendocrine reproductive events. 79

92 CHAPTER 5: Kisspeptin and GnIH-mediated regulation of GnRH mrna levels in a Novel GnRHsecreting Cell Model 80

93 5.1 Abstract Reproduction is a complex and highly coordinated event, which ensures that the internal environment is optimal for successful procreation. GnRH neurons, the integrators of the HPG axis, largely facilitate reproductive functions. Kisspeptin has emerged as a potent stimulator of GnRH neuronal activity in the hypothalamus. Another hypothalamic neuropeptide was identified, GnIH, and was found to inhibit gonadotropin synthesis and secretion at the hypothalamic and pituitary levels. To date, there is a paucity of studies focusing on the regulatory effects of kisspeptin and GnIH peptides on GnRH neurons. To address this issue, we have examined the effect of kisspeptin on GnRH gene expression in a novel GnRH neuronal cell model, mhypoa- GnRH/GFP, expressing the kisspeptin and GnIH receptors (Gpr54 and Gpr147 respectively). Incubation of mhypoa-gnrh/gfp neurons with 10 nm of Kiss-10 stimulated c-fos expression, and induced GnRH mrna expression at 4 h. We also evaluated the combined effect of stimulatory and inhibitory regulators of GnRH neuronal activity by performing a co-treatment of Kiss-10 (10 nm) and GnIH (100 nm). We report an overall attenuation of GnRH mrna expression transiently following co-treatment up to 2 h following treatment. This suggests that GnIH induces GnRH neuronal suppression more rapidly than kisspeptin-mediated induction. Using the mhypoa-gnrh/gfp cell line, our studies indicate a central mechanism for kisspeptinmediated up-regulation of GnRH mrna expression. Furthermore, the suppressive signaling of GnIH peptide on GnRH neurons can reverse the stimulatory effect of kisspeptin. Thus, we demonstrate the integration of stimulatory and inhibitory afferents by a novel GnRH cell model. 5.2 Introduction The functioning of the hypothalamic-pituitary-gonadal (HPG) axis, like all other endocrine systems, is dependent on vastly interconnected networks of communicating neuronal and organ systems. Located at the pinnacle position of the rodent HPG axis is the anterior 81

94 hypothalamus, where a small population of neurons, are responsible for synthesizing and secreting the decapeptide GnRH [2-4, 24]. The population of GnRH neurons function as the central integrator and ultimate effector for the different modulators of both puberty onset and all subsequent reproductive functions throughout life [4]. GnRH is released from the nerve terminals at the median eminence as timed, highly synchronized secretory bursts [25] [26] and is released into the hypothalamic-hypophyseal portal system, where it is carried to the adenohypophysis [20, 27]. GnRH stimulates gonadotrope cells of the anterior pituitary to synthesize and secrete pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) into portal circulation in a pulsatile fashion, following a similar time frame as GnRH secretion [29]. Finally the gonads, which, in addition to initiating gametogenesis at puberty, respond to the trophic actions of gonadotropins by escalating sex-steroid and peptide hormone secretion [298]. In the HPG axis, the fluctuating pulse frequency of GnRH release is the chief mechanism by which the body is able to modify reproductive status during maturation and developmental processes such as puberty [29]. Several regulators of the HPG axis have been identified, including gonadal steroids, neurotransmitter and neuropeptide systems, metabolic signals and environmental cues. Our understanding of the neurohormonal basis for the activation of the HPG axis has undergone substantial conceptual amendments, with emphasis now being placed on changes in the sensitivity to the feedback effects of sex steroids, and on modifications in the ratio between central inhibitory and stimulatory inputs to the GnRH neurons. On the latter, it is now globally accepted that the activation of GnRH neurons is brought about by the concomitant decrease in the restrain of inhibitory afferents and the increase in stimulatory inputs [116]. Furthermore, while various stimulatory environmental signals had been elucidated upstream of the GnRH neurons, the stimulatory neuronal systems upstream remained more ambiguous until the role of kisspeptin in reproductive physiology was realized [10, 180, 181]. Kisspeptins are a family of neuropeptides, belonging to the family of RF-amide peptides, and are encoded by the Kiss-1 gene 82

95 [14]. Kisspeptins act via binding to their cognate G protein-coupled receptor (GPCR), Gpr54, thus mediating an essential role as the potent activator and major gatekeeper of the HPG axis [14, 16, 17]. Kisspeptin and Gpr54 are recognized as critical players in the control of key aspects of reproductive function, ranging from neonatal sexual differentiation, to regulation of GnRH and gonadotropin secretion, to the metabolic gating of puberty and fertility. Kisspeptin has since demonstrated its potent stimulatory role in the reproductive axis, displaying the ability to induce GnRH secretion and the resulting LH pulse in several species, including rodents, sheep, primates and humans [ ]. In addition, the Kiss1-Gpr54 system is responsible for integration of more upstream signals from steroid hormones [183, 184], and is subject to regulation by metabolic factors [250, 256], further supporting their key role as gatekeepers of GnRH neurons. While in vivo studies of the Kiss1-Gpr54 system are plentiful, there exists a paucity of in vitro studies, namely due to the complexity and herterogenity of the hypothalamus, where major kisspeptin populations have been localized. In 2000 Tsutsui and colleagues discovered a novel hypothalamic dodecapeptide isolated from the quail brain, with the ability to inhibit gonadotropin release from the anterior pituitary [150], becoming the first hypothalamic peptide known to inhibit gonadotropin release in vertebrates. Appropriately, the peptide was named gonadotropin-inhibitory hormone (GnIH) and has been localized to cell bodies in the PVN and to terminals in the ME [150]. The GnIH receptor has been identified as a novel G-protein coupled receptor (Gpr147), binding GnIH peptide in a concentration-dependent manner, and its mrna has been found in the pituitary and the diencephalon [153]. These results suggest that GnIH acts to inhibit gonadotropin release directly by binding to the GnIH receptor at the pituitary [152, 153]. Subsequent in situ hybridization studies revealed expression of Gpr147 mrna in GnRH neurons [263], suggesting that GnIH also acts in the hypothalamus to affect GnRH neuronal activity. To date, the majority of mammalian studies have focused on the role of kisspeptin and GnIH as hypophysiotropic factors stimulating/inhibiting GnRH-induced gonadotropin secretion 83

96 from the pituitary. The molecular mechanisms that regulate both kisspeptin and GnIH-mediated changes in GnRH synthesis at the level of the hypothalamus however, remain unexplored. For this reason, the present study aims to elucidate the effects of kisspeptin and a kisspeptin/gnih cotreatment on GnRH synthesis. To complete these studies, we have established a novel non-clonal hypothalamic GnRH neuronal cell model, the mhypoa-gnrh/gfp neurons, demonstrating GnRH secretion, expression of Gpr54 and Gpr147 (McFadden et al., manuscript submitted; Gojska et al., manuscript submitted). For the first time, here we report that that kisspeptin can act on hypothalamic GnRH neurons in vitro increasing GnRH biosynthesis, and furthermore, that following a co-treatment of kisspeptin and GnIH, GnRH biosynthesis is transiently suppressed. 5.3 Results Characterization of Gpr54 and Gpr147 mrna expression in mhypoa-gnrh/gfp neurons We have previously reported the generation and characterization a novel non-clonal, adultderived GnRH-synthesizing hypothalamic cell line (McFadden et al., manuscript submitted). In brief, the cell line was created following isolation of primary hypothalamic tissue from a 2- month-old transgenic GnRH-GFP mouse. The tissue was immortalized using retroviral transfection methods, specifically, the simian virus 40 T antigen containing a neomycin resistance gene, and selecting for transfected neurons using geneticin. Subsequently, we performed fluorescence activated cell sorting (FACS), and a non-clonal GnRH model was generated using a previously established protocol. Prior work has demonstrated expression of estrogen receptor (ER) genes in the mhypoa-gnrh/gfp model (McFadden et al., manuscript submitted). For the purpose of better characterizing the novel cell line, we performed RT-PCR to determine if our GnRH neuronal model also expresses Gpr54 and Gpr147. Results confirm expression of mrna 84

97 A M Hypo mhypoa-gnrh/gfp NTC Gpr147 B M Hypo mhypoa-gnrh/gfp NTC Gpr54 Figure 5.1. Gene expression profile of an adult-derived, non-clonal GnRH-secreting cell line, mhypoa-gnrh/gfp Representative RT-PCR screening of Gpr147 (A) and Gpr54 (B). In brief, total RNA was isolated and amplified using the One-Step RT-PCR Qiagen kit with genespecific primers. Products were visualized on a 2% agarose gel with whole hypothalamus (positive control) and a non-template control (NTC; negative control). 85