La reproduction dans des mammifères est réglée par les neurones qui synthétisent et sécrètent l'hormone de gonadotropin-sortie (GnRH) et à travers l'espèce ces neurones sont présents dans peu de nombres dispersés dans l'hypothalamus. En raison de limité neurogenesis de ceux des types cellulaires neuronaux à l'extérieur du cerveau dans placode olfactif, ces neurones sont soumis au règlement serré pendant le développement embryonnaire pour atteindre leurs objectifs finaux dans l'hypothalamus, de la naissance jusqu'à la puberté pour la sécrétion minimale d'hormone et pendant des adultes pour réaliser la sécrétion pulsatile de l'hormone. La dérégulation dans n'importe lequel de ces mécanismes peut mener aux effets délétères sur la reproduction adulte et des pathologies cliniques comme l'absence de puberté, hypogonadism, la stérilité, l'aménorrhée, etc. Kallmann syndrome (KS), un d'entre ceux sévères (graves) reproducteur / Reproduction in mammals is regulated by neurons that synthesize and secrete gonadotropin-releasing hormone (GnRH) and across the species these neurons are present in few numbers scattered in the hypothalamus. Due to limited neurogenesis of these neuronal cell types outside the brain in the olfactory placode, these neurons are subjected to tight regulation during embryonic development to reach their final targets in the hypothalamus, from birth until puberty for minimal secretion of hormone and during adults to achieve pulsatile secretion of the hormone. Deregulation in any of these mechanisms may lead to deleterious effects on adult reproduction and clinical pathologies like absence of puberty, hypogonadism, sterility, amenorrhea, etc. Kallmann syndrome (KS), one of these severe reproductive pathologies is an inherited disorder and patients affected with this syndrome display anosmia (inability to smell) and hypogonadotropic hypogonadism (HH). Genetic screening of molecules in these patients lead to identification of genes like KAL1, FGFR1, FGF8, PROK2, PROKR2, WDR11 and CHD7 encoding proteins that play an important role in migration and targeting of olfactory system during embryonic development however these genes account only for 30% of KS cases emphasizing the need for further characterization and identification of other genes. While these proteins are involved in ontogenesis olfactory and GnRH system, genetic screening of molecules in patients suffering from normosmic idiopathic HH lead to identification of genes encoding for Kisspeptin receptor-GPR54, TAC-TACR3, LEP-LEPR, PCSK-1, GnRH receptor-GnRHR and GnRH-1 itself that play a crucial role in occurrence of puberty or adult reproduction. Here, for my PhD thesis, we focused on studying the role of guidance molecule Semaphorin3A (Sema3A)-Neuropilin1 (Nrp1) interactions in ontogenesis of GnRH neurons during embryonic development while in adults we first addressed the question if hypothalamic Kisspeptin neurons interact with neurons containing neuronal nitric oxide synthase (nNOS), the mutation of which causes HH in mice, and physiological significance of this interactions in regulation of GnRH neurons and neuroendocrine control of female reproduction. Finally our results demonstrate that Sema3A-Nrp1 interactions are implicated in ontogenesis of olfactory and GnRH neurons during embryonic development and nNOS neurons are important mediators of peripheral estrogens-kisspeptin signaling onto GnRH neurons and adult reproduction and propose to further study the implication of nNOS neurons in reproductive pathologies.

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.