Increased pulsatile release of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) is critical for pubertal initiation of ovarian cycles in female mammals. Kisspeptin, a peptide product of the Kiss1 gene, is required for normal puberty. In Experiment 1, ovariectomized ewe lambs bearing subcutaneous estradiol implants were used to investigate Kiss1 gene expression in the preoptic area (POA) and hypothalamus during pubertal maturation of the reproductive neuroendocrine system. Brain tissue was collected from ewes at 25, 30, and 35 wk of age (n=6/group). Patterns of LH release in circulation were determined on the day before euthanasia and cells containing Kiss1-mRNA were identified by in situ hybridization. Mean concentrations of LH and the frequency of LH pulses increased (P < 0.01) as ewe lambs matured. In the POA/Periventricular area (PEV), the number of Kiss1-expressing cells was greater (P < 0.04) in 30- and 35-wk-old than in 25-wk-old ewe lambs. In the arcuate nucleus (ARC), although no significant changes in number Kiss1-expressing cells were observed among age groups, the number of Kiss1 cells increased (P < 0.02) with increased frequency of LH release. This resulted in greater (P < 0.01) number of Kiss1 cells in the ARC of ewes demonstrating elevated frequency (> 6 pulses/12 h) of LH pulses. In Experiment 2, 28-wk-old ewe lambs were used to determine the effects of intermittent injections of kisspeptin on the release of LH and stimulation of gonadal function in peripubertal ewe lambs. Ewe lambs were treated intravenously with saline (Controls; n=6) or kisspeptin (n=6) hourly for 24 h. Blood samples were collected throughout the experiment for hormone analysis. Kisspeptin-treated lambs had greater (P < 0.02) mean circulating concentrations of LH, and frequency and amplitude of LH pulses than controls. Four of six kisspeptin-treated ewe lambs exhibited LH surge and luteal activity in response to treatments. However, onset of regular estrous cycles was not established immediately following kisspeptin-induced ovulation and no difference in age at onset of puberty was observed between groups. In conclusion, activation of the hypothalamic kisspeptin system may support elevated episodic release of LH critical for establishment of normal estrous cycle during pubertal development.

Thesis--Auburn University, 2008. / Abstract. Vita. Includes bibliographical references (p. 43-50).

Context: The human foetal adrenal (HFA) is an integral component of the foeto-placental unit and important for the maintenance of pregnancy. Low kisspeptin levels during pregnancy are associated with miscarriage, and kisspeptin and its receptor are expressed in the HFA. However, the role of kisspeptin in foetal adrenal function remains unknown. Objective: The objective of this work was to determine the role of kisspeptin signaling in the developing HFA. Methods: Experiments using H295R adrenocortical cells and primary HFA cells as in vitro models of the foetal adrenal were conducted. The spatiotemporal expression of the kisspeptin receptor, Kiss1R in the HFA was examined. The production of dehydroepiandrosterone sulphate (DHEAS) from HFA cells after kisspeptin treatment, alone or in combination with adrenocorticotropic hormone or corticotropin-releasing hormone was compared. The association of plasma kisspeptin levels with HFA size was analysed in a longitudinal clinical study. Thirty-three healthy pregnant women were recruited at their 12-week routine antenatal ultrasound scan. Serial measurements of foetal adrenal volume (FAV) and maternal kisspeptin levels at four antenatal visits (∼20, 28, 34, and 38 weeks' gestation) were collected. Outcomes of pregnancy were recorded, including neonatal auxology and complications in the postnatal period. Neoantal adrenal remodeling was characterized by Results: Expression of Kiss1R was present in the HFA from 8 weeks after conception to term and was shown in the inner foetal zone. Kisspeptin significantly increased DHEAS production in H295R and second-trimester HFA cells. Serial measurements of kisspeptin confirmed a correlation with FAV growth in the second trimester, independent of sex or estimated foetal weight. Conclusions: Together, these studies demonstrate that kisspeptin plays a key role in the regulation of the HFA, and thus the foeto-placental unit, particularly in the second trimester of pregnancy.

In the United States, there are currently more than 40 million postmenopausal women. These women are faced with a variety of physiological changes including ovarian steroid withdrawal and alterations in hypothalamic neurons. Within the hypothalamic infundibular nucleus of postmenopausal women, there is neuronal hypertrophy and an increase in neurokinin B gene expression. Recent studies identified the kisspeptins and dynorphins as major regulators of reproduction. In our first experiment, we examined the location and alterations of KiSS-1 mRNA-expressing neurons in the hypothalami of pre and postmenopausal women. KiSS-1 neurons were largely confined to the infundibular nucleus, and in postmenopausal women, exhibited neuronal hypertrophy and increased gene expression. To determine if these changes could result from alterations in ovarian steroids, we investigated KiSS-1 gene expression in the hypothalamic infundibular nucleus of non-human primates. Similar to the findings in postmenopausal women, ovariectomy of monkeys resulted in neuronal hypertrophy and increased KiSS-1 gene expression within the infundibular nucleus. Further, estrogen treatment of ovariectomized monkeys yielded a dramatic decrease in KiSS-1 gene expression. Together, these findings suggest that the postmenopausal alterations in KiSS-1 neurons are secondary to ovarian failure.In a second study, we examined alterations in dynorphin gene expression in the hypothalami of pre and postmenopausal women. Dynorphin mRNA-expressing neurons were identified in multiple nuclei. Numbers of dynorphin neurons were decreased within the mPOA and infundibular nucleus of postmenopausal women. In the infundibular nucleus of postmenopausal women, dynorphin neurons were hypertrophied. To determine the contribution of ovarian steroids on dynorphin gene expression, we examined dynorphin mRNA in a monkey model of menopause. Young ovariectomized monkeys exhibited hypertrophy of dynorphin neurons, with no changes in dynorphin gene expression. Estrogen replacement yielded a decrease in neuronal size and an increase in dynorphin neuron number.In future studies, we will use Quantum Dot FISH to determine if NKB, KiSS-1, and dynorphin are colocalized in the hypertrophied neurons. These neuropeptides are involved in the regulation of GnRH and changes in their gene expression likely contribute to postmenopausal alterations in reproductive hormones. Our findings provide greater understanding of the postmenopausal condition and offer opportunities for pharmaceutical investigation and treatment.

Thesis (Ph. D.)--Auburn University. / Abstract. Vita. Includes bibliographical references (p. 176-225).

Preeclampsia is an increasingly prevalent disorder of placentation that has very limited options for treatment. The disease is characterized by aberrant invasion of placental trophoblasts into the decidualized maternal endometrium. In order to identify pathways of therapeutic interest during placentation, we are focusing on the pathway of the neuropeptide kisspeptin and its receptor KISS1R, both highly expressed in the human placenta. Early functional studies of the ligand-receptor system identified a role for kisspeptin in the inhibition of cancer metastasis. Parallels exist between cancer and placentation, suggesting the possibility of an inhibitory role for kisspeptin during pregnancy as well. Existing functional data supports kisspeptin's inhibitory influence on cellular invasion, but the mechanism remains unknown. Evidence for the localization of the KISS1R receptor in the current literature was established via a nonspecific antibody and requires further investigation. Current literature suggests involvement of the ERK (extracellular signal-regulated kinase) pathway as well. Our work aims to solidify the localization of kisspeptin and KISS1R, avoiding the use of KISS1R antibodies. Using immunohistochemistry for protein localization of kisspeptin and placental fractionation followed by quantitative PCR analysis for gene expression, we provide evidence of kisspeptin's restriction to the syncytiotrophoblast layer of the placenta, and KISS1R gene expression limited to the villous cytotrophoblast layer. This distribution of ligand and receptor suggests a paracrine mechanism for kisspeptin action, with syncytiotrophoblasts secreting kisspeptin to act on its receptor on the villous cytotrophoblast layer, and thus restricting cytotrophoblast invasion. We further attempt to support these data with the use of laser capture microdissection of placental tissue to isolate the different layers, followed by quantitative PCR. This technique introduced a particularly challenging aspect of working with the placenta: maintaining tissue morphology while also preserving RNA integrity. This thesis outlines our troubleshooting process for that technique and introduces alternatives for future work. We also employed Western blot analysis of ERK activation to establish the mechanism of kisspeptin's inhibitory effect on fractionated trophoblasts.

Stressors generate adaptive responses, including transient suppression of reproductive function. Natural selection depends on successful reproduction, but inhibition of reproduction to survive famine or escape predation allows animals to survive to reproduce at a later time. The cellular locations and mechanisms responsible for inhibiting and reactivating the reproductive axis during and after stress, respectively, are not well understood. We demonstrated that stress-induced elevation in glucocorticoids affects hypothalamic neurons that secrete kisspeptin (KISS1), an important reproductive hormone. Stressors that stimulated glucocorticoid secretion, as well as glucocorticoid administration itself, inhibited Kiss1 mRNA expression, while conditions that did not change glucocorticoid secretion did not alter Kiss1 mRNA expression. In mice lacking glucocorticoid receptor specifically in kisspeptin-containing neurons, Kiss1 mRNA expression was no longer inhibited during restraint stress despite a rise in corticosterone, and both testosterone and copulatory behaviors showed accelerated recovery in the post-traumatic period. We also demonstrated that increased glucocorticoid secretion contributed to infertility and obesity in leptin-deficient mice. Leptin deficiency creates a chronic state of perceived starvation, and leptin-deficient mice exhibit elevated plasma glucocorticoid concentrations, morbid obesity, and infertility. Leptin-deficient, glucocorticoid-deficient mice exhibited decreased body weight and fat composition, decreased hyperphagia, and normal fertility. When supplemented with glucocorticoids back to the initial levels present in leptin deficiency, these mice gained weight and became infertile. Thus, leptin is not required for fertility as previously believed, and glucocorticoids can contribute to obesity and suppress fertility independently of leptin signaling. Together, these findings implicate glucocorticoids in the regulation of obesity and reproductive inhibition during stress, including perceived starvation caused by leptin deficiency. These studies may provide novel mechanisms and molecular targets in the reproductive and metabolic aspects of disorders characterized by glucocorticoid dysregulation, including post-traumatic stress disorder, anorexia nervosa, and mood disorders.

Le kisspeptine (kp) est un peptide, dérivé du gène kiss-1, jouant un rôle majeur dans le contrôle central de la fonction de reproduction en régulant la sécrétion du GnRH chez l’adulte mais également au cours du développement. Les neurones exprimant kiss-1 sont situés dans la région rostrale périventriculaire du 3ème ventricule (RP3V) et le noyau arqué (ARC). L’expression de kiss-1 est hautement régulée par les stéroides sexuels, positivement dans RP3V et négativement dans ARC. Ces deux populations de neurones à kp semblent avoir des rôles différents. Les neurones à kp du RP3V seraient impliqués dans la genèse du pic préovulatoire et ceux de l’ARC dans la régulation de la sécrétion pulsatile de GnRH.L’objectif de la thèse était de déterminer la période de neurogenèse des neurones à kp ainsi que les variations de l’expression de kiss-1 et de kp dans ces deux régions au cours des différentes phases du développement chez le rat mâle et femelle.Nos résultats ont permis de cibler les périodes clés de l’ontogenèse des neurones à kp en montrant 1) que les neurones à kp de l’ARC naissent sur une période étendue à partir du jour embryonnaire (E)12,5; 2) l’existence d’une sous- expression péri-natale du kp dans l’ARC indépendante du sexe; 3) la mise en place, en période néonatale, de différences sexuelles dans les niveaux d’expression et la distribution neuroanatomique du kp; 4) l’existence de régulations péri-pubertaires de kp, dépendantes du sexe et de la région ; 5) la présence de fibres à kp dans des régions hypothalamiques suggère un rôle de kp au-delà de la fonction de reproduction. / Kisspeptin (kp) is a neuropeptide, derived from the kiss-1 gene, which plays a key role in the central control of reproduction by regulating GnRH secretion in adult but also during development. Cells which express kiss-1 are localized in two distincts hypothalamic regions: the rostral peri-ventricular third ventricule area (RP3V) and the arcuate nucleus (ARC). Kiss-1 expression is highly regulated by sex steroids: positively in the RP3V and negatively in the ARC. RP3V kp neurons have been implicated in the pre-ovulatory GnRH surge whereas ARC kp neurons may predominantly act on GnRH secretion pulsatility. The aim of this PhD work was to determine the neurogenesis period of kp neurons and changes of kiss-1 and kp expression in both regions during different stages of development in rats. Our results highlight key periods of kp neurons ontogenesis and show that: 1) ARC kp neurons are born during an extended embryonic neurogenesis period starting at embryonic day (E) 12,5; 2) a sex independent down-regulation of kp occurs during peri-natal period; 3) sex difference in the expression level and neuroanatomique distribution of kp establishes during neo-natal period; 4) kp was regulated during peri-pubertal period in sex and region dependant manner; 5) kp-ir fibers are detected throughout the septo-hypothalamic continuum suggesting that kp could be implicated in other functions than reproductive function.

Background: Kisspeptins, recently discovered hypothalamic neuropeptides encoded by the KISS1 gene, are essential for normal pubertal development and are modulated by diverse endocrine, metabolic and environmental signals. Exogenous kisspeptin administration potently stimulates LH secretion - by direct action on GnRH neurons while kisspeptin antagonists inhibit pulsatile LH secretion. Human studies of kisspeptin had hitherto used kisspeptin-54 that is cleaved further and the smallest bioactive form is a decapeptide (kisspeptin-10) with a shorter half-life. Kisspeptin-10 is thus putatively more attractive in studies assessing LH pulsatility and is also the basis for the development of antagonists. Unmet clinical needs: Decreased LH pulse frequency is the central pathology in pubertal delay, late-onset male hypogonadism and hypothalamic amenorrhoea. Manipulation of LH pulse frequency also has therapeutic potential in contraception, PCOS and sex-steroid dependant diseases such as endometriosis and prostatic hyperplasia. Hypothesis: That exogenous kisspeptin-10 enhances pulsatile LH secretion in healthy men and in patients with reproductive disorders associated with decreased pulse frequency. Research strategy: A first-in-human dose escalation study of kisspeptin-10 was performed in men and subsequently replicated in women. An intravenous infusion regime was optimised in healthy men and subsequently applied to hypogonadal patients. Specific questions were addressed sequentially as summarised below with key results. Dose escalation study: Question: Does kisspeptin-10 stimulate LH secretion in men? Findings: Six iv bolus doses (0.01 to 3 μg/kg) of GMP kisspeptin-10 and vehicle were administered at least a week apart to six healthy men. Rapid increase in LH, with peak concentrations was seen by 45 min post injection in all volunteers. There was a clear dose-dependent increase in LH concentrations in response to kisspeptin- 10 (P <0.0001). Area-Under-Curve analysis over 60 min following kisspeptin-10 administration showed 0.3 and 1μg/kg doses to be maximally stimulatory (P <0.01) with a reduced response at 3 μg/kg. Assessing the effect of steroid milieu: Question: Steroid feedback is central to the regulation of LH secretion: what effect does the steroid milieu have on LH responses to kisspeptin-10? Findings: The response to iv kisspeptin-10 (0.3μg/kg,) in the normal follicular phase (n=10) was compared with that in the presence of low endogenous sex steroids/high LH secretion (6 postmenopausal women) and in women taking combined contraceptive therapy (n=8) with suppressed LH secretion. Despite widely varying baseline secretion, LH increased significantly following kisspeptin-10 administration in the follicular phase (6.3±1.2 to 9.4±1.3 IU/L P=0.006), postmenopausal (35.3±2.8 to 44.7±3.4 IU/L P=0.005), etonogestrel (4.6±0.2 to 7.5±0.9 IU/L, P=0.02), and COCP groups (2.2±0.9 to 3.7±1.4 IU/L P<0.001). Pulse frequency study: Question: GnRH and LH secretion are pulsatile: can kisspeptin-10 enhance LH pulsatility? Findings: Four healthy men attended our clinical research facility for two visits five days apart for 10-min blood sampling. At the first visit, baseline LH pulsatility was assessed over a 9-hour period. During the second visit, an infusion of kisspeptin-10 was administered for 9 hours at 1.5μg/kg/hr after an hour of baseline sampling. LH pulse frequency increased in all subjects, with a mean increase from 0.7±0.1 to 1.0±0.2 pulses/hr (P = 0.01), with resultant increase in mean LH from 5.2±0.8 IU/L at baseline to 14.1±1.7 IU/L (P <0.01). High dose, longer duration infusion study: Question: Can kisspeptin-10 enhance testosterone secretion? Findings: Four healthy men attended our clinical research facility for a 34-hour supervised stay. Blood samples were collected at 10 min intervals for two 12 hour periods on consecutive days and hourly overnight. After 10.5 hours of baseline sampling a continuous intravenous infusion of kisspeptin-10 (4μg/kg/hr) was maintained for 22.5 hrs. Mean LH increased from 5.5±0.8 at baseline to 20.9±4.9 IU/L (P <0.05) and serum testosterone increased from 16.6±2.4 to 24.0±2.5 nmol/L (P <0.001). Translational studies in hypogonadal men with type 2 diabetes Question: Can kisspeptin-10 normalise testosterone secretion in hypogonadal men? Findings: Five hypogonadal men with T2DM (age 33.6±3 yrs, BMI 40.6±6.3, testosterone 8.5±1.0 nmol/L, LH 4.7±0.7 IU/L, HbA1c <8 %, duration of diabetes <5 yrs) and seven age matched healthy men were studied. Kisspeptin-10 was administered intravenous (0.3 μg/kg) with frequent (10-min) blood sampling. Mean LH increased in controls (5.5±0.8 to 13.9±1.7 IU/L P <0.001) and in T2DM (4.7±0.7 to 10.7±1.2 IU/L P=0.02) with comparable ΔLH (P=0.18). Baseline serum sampling for LH at 10-min intervals and hourly testosterone measurements were performed subsequently in four T2DM men for 12 hours. An intravenous infusion of kisspeptin-10 (4 μg/kg/hr) was administered 5 days later for 11 hours, with increases in serum LH (3.9±0.1 IU/L to 20.7±1.1 IU/L (P=0.03,) and testosterone (8.5±1.0 to 11.4±0.9 nmol/L, P=0.002). LH pulse frequency at baseline was lower in hypogonadal men with diabetes (0.6±0.1 vs. 0.8±0.1 pulses/hr, P=0.03) and increased to 0.9±0 pulses/hr (P=0.05). Translational studies in pubertal delay: Question: Defective Neurokinin B activity is associated with pubertal delay and the hierarchical interactions between kisspeptins and Neurokinin B remain to be elucidated: can kisspeptin-10 stimulate LH secretion with impaired Neurokinin B signalling? Findings: Four patients with TAC3 or TACR3 inactivating mutations presenting with delayed puberty were admitted for two 12 hr blocks of blood sampling every 10 min with vehicle (saline) or kisspeptin-10 (1.5 μg/kg/hour) infused intravenously. Mean LH and LH pulses frequency increased with kisspeptin-10 (P<0.05). However, four patients with Kallmann syndrome (with defective GnRH neuron migration), studied in parallel, did not respond, suggesting a potential diagnostic application for kisspeptin-10 in pubertal dysfunction. Conclusions In first-in-man studies of kisspeptin-10, it was demonstrated that endogenous LH pulse frequency can be enhanced in healthy men. The therapeutic potential of this finding in common reproductive endocrine disorders associated with decreased LH pulse frequency, i.e., late-onset male hypogonadism and pubertal dysfunction, was suggested in subsequent studies. Furthermore, kisspeptin signalling occurs upstream of GnRH neurons and is independent of Neurokinin B signalling in the central regulation of the hypothalamic-pituitary-gonadal axis.

GnRH neurons represent the final common pathway for the regulation of the reproductive axis and they are modulated by multiple signals. It has recently been shown that a potent effector of GnRH neuron function is an afferent network of kisspeptin-producing neurons. Kisspeptin released from these neurons acts upon a specific receptor (gpr-54) expressed on GnRH neurons, and increases the secretion of GnRH from the hypothalamus. The kisspeptin system has since been implicated as a downstream mediator for regulation of the Hypothalamic-Pituitary-Gonadal (HPG) axis by steroid hormones, metabolic signals and photoperiod, potentially placing it at the centre of reproductive physiology. However, the supporting evidence to date has been indirect, relying on interpretation of changes in mRNA levels and immuno-histochemical staining to infer the actions of kisspeptin upon the central control of reproduction. The detailed mechanisms of kisspeptin action are yet to be fully elucidated. The research within this thesis elucidates the effect of kisspeptin on the HPG axis via the development of kisspeptin-10 (kp-10) analogues with antagonistic properties. Functionally important residues within the peptide were delineated. Structure-activity studies of kp-10 analogues indicated that residues Asn2, Trp3, Phe6, Arg9 and Phe10 interact with gpr-54 to facilitate receptor binding. Two other residues, Tyr1 and Leu8 were shown to be critical for receptor activation by kisspeptin. Four synthetic peptide antagonists were selected according to a consensus sequence for good antagonism: X1-N-W-N-X5-F-G-X8-R-F-NH2 where X1 = D-Ala or D-Tyr, X5 = Gly or D-Ser and X8 = D-Trp or D-Leu. One of the antagonists, peptide 234, was used in in vivo studies, where it inhibited the amplitude of GnRH and LH pulses without affecting basal secretion of GnRH or LH. These results indicate for the first time that basal and pulsatile secretion of these factors is regulated by separate pathways. Use of the antagonist also demonstrated the direct involvement of endogenous kisspeptin in steroid hormone negative feedback, positive regulation of the pre-ovulatory LH surge and in regulating the onset of puberty in rodents, as had been suggested via indirect methods. Although a major role of the kisspeptin system is in the regulation of the HPG axis, the system may also be important in the inhibition of cancer cell metastasis and in placental development (trophoblast cell invasion) but little is known about the mechanisms involving kisspeptin in these processes. This thesis describes novel signalling mechanisms for the regulation cell migration by kisspeptin, involving the MAPK and GSK3β signalling pathways. Using a stably transfected CHO cell line, kisspeptin-gpr-54 signalling can activate all members of the MAPK pathway, the β- catenin/GSK3β pathway, NFκB and FAK. These factors are involved in inhibiting the migration of these cells via an ERK1/2-p90rsk-GSK3β-β catenin pathway to potentially up- regulate formation of adherens junctions at the plasma membrane. This pathway was also shown to be involved in the inhibition of migration within an immortalised human first trimester placental trophoblast cell line and in human umbilical vein endothelial cells. Some of these pathways were also active within a mouse GnRH neuronal cell line, where ERK1/2, NFκB and GSK3β were activated by kisspeptin with no effect on migration. However, the role of these pathways in the GnRH neuronal cells requires further investigation. In summary, the research presented within this thesis defines receptor-binding and activating residues within kisspeptin-10, which should enable more details of ligand-receptor binding interactions to be fully elucidated. Novel gpr-54 antagonists have been identified and used in in vivo studies. The thesis demonstrates the direct involvement of endogenous kisspeptin in the regulation of GnRH/LH secretion at the onset of puberty and throughout the reproductive cycle in mature animals. The antagonists developed within this thesis represent useful tools to further delineate mechanisms of kisspeptin action within the HPG axis and peripheral tissues. Other findings describe kisspeptin signalling mechanisms for the inhibition of cell migration, potentially important in a variety of normal and pathological processes, including for the first time a description of the regulation of GSK3β and β-catenin signalling factors by kisspeptin and gpr-54.