Neural control of reproduction in vertebrates and invertebrates has generated considerable interest due to the presence of common neuropeptides. Gonadotropin-releasing hormone (GnRH), a neuropeptide, is the final integrator of neural regulation governing reproduction in vertebrates by controlling the release of gonadotropins. Little is known about GnRH before the origin of vertebrates or about the biological significance of multiple GnRH forms in a single species. To understand the role of GnRH in invertebrates, I selected a tunicate, Ciona intestinalis, the sister group to vertebrates and amphioxus, Branchiostoina floridae, a group more basal than tunicates. Neural control of reproduction in these chordates was compared with that in the zebrafish, Danio rerio. From the zebrafish, I isolated four GnRH receptor cDNAs that each map to a distinct chromosome and are expressed in a variety of tissues. Each receptor was functional, as shown by its response to physiological doses of native GnRH peptides. Also, two receptors showed selectivity between GnRH1 and GnRH2. Protein localization of each zebrafish GnRH receptor with specific antisera showed that all four receptors are present in the pituitary. However, the most striking localization revealed the presence of GnRH networks in a major motor control centre and fibre tract system in the hindbrain and spinal cord. Both structures are major components in the control of motor movements, such as swimming. Phylogenetic and synteny analysis segregates the four zebrafish GnRH receptors into two distinct phylogenetic groups that are separate gene lineages conserved throughout vertebrate evolution. In Ciona intestinalis, we found two GnRH genes that each encode three GnRH decapeptides in tandem, for six unique GnRH forms from this species. These genes are expressed throughout development. With an immunocytochemical approach, at least one peptide was found in the dorsal strand nerve plexus adjacent to the gonads in adults. Injection near the gonads of gravid Ciona quickly induced spawning, suggesting a novel action for control of reproduction by GnRH. My further studies identified four novel GnRH receptors encoded within the genome of this protochordate, and showed that three receptors responded to Ciona GnRHs by stimulating intracellular accumulation of cAMP. In contrast, only one receptor activated inositol phosphate turnover in response to one of the Ciona GnRHs. My final study involved identifying the GnRH signalling components in amphioxus. I found four novel GnRH receptors, with three displaying sensitivity to the highly conserved vertebrate GnRH2 and one of these showing selectivity for GnRH1. My pharmacological testing showed that the capacity to respond to GnRH1 and GnRH2 is evolutionarily conserved between amphioxus and vertebrates, and that key motifs found to be important in GnRH binding, signalling and activation are present in the amphioxus receptors. Phylogenetic analysis showed that two receptors cluster with the recently identified octopus GnRHR-like sequence; the other two receptors group at the base of the vertebrate GnRHR clade and may represent the proto-vertebrate condition, after which gene duplication and sequence divergence resulted in the four contemporary vertebrate GnRHRs. This work reveals novel and important features of the GnRH signalling axis throughout chordate evolution.

[Truncated abstract] It is becoming increasingly obvious that cell signalling pathways are more complicated than we originally perceived. Research is revealing that, not only is there a multitude of new proteins involved in signalling cascades, but also that previously identified proteins may have additional, alternate roles in intracellular trafficking. Gonadotropin-releasing hormone (GnRH) in conjunction with its receptor (GnRHR), the primary regulator of reproduction in all species, is no exception. In the past few years it has become readily accepted that the classic linear GnRHR-Gαq/11 signalling pathway is not universal and that this receptor is involved in a far greater range of cellular activities than was previously considered. In particular, it is widely accepted that continuous administration of GnRH analogs results in an inhibition of growth of a number of reproductive-derived tumours and that this may, in part, be mediated by direct activation of GnRHs expressed on these cells. However, it is not fully understood how the GnRHR mediates these growth effects or whether such effects are unique to reproductive-derived cancer cells. Research within this thesis aimed to determine how the presence or absence of this receptor in different cell types might affect the ability of GnRH to directly mediate growth effects. We demonstrate that continuous treatment with a GnRH agonist (GnRHA) induces an anti-proliferative effect in a gonadotropederived cell line (LβT2) and also in HEK293 cells stably expressing either the rat or human GnRHR. The anti-proliferative effect was time- and dose-dependent and was specifically mediated via the GnRHR, as co-treatment of the GnRHRexpressing cell lines with a GnRH antagonist blocked the growth suppressive effect induced by GnRHA treatment. Cell cycle analysis revealed that the GnRHA treated HEK/GnRHR cell lines induced an accumulation of cells in the G2/M phase while a G0/G1 arrest was observed in LβT2 cells. Previous identification by our group of a potential interaction between the GnRHR and the transcription factor E2F4, an integral cell cycle regulatory protein, prompted further investigation as to the nature of this interaction. Bioluminescence energy transfer (BRET) was utilised to demonstrate that the GnRHR also interacts with E2F5, another member of the E2F family of cell cycle proteins that shares a high level of homology to E2F4. In addition, it was determined that the interaction between human GnRHR and E2F4, detected using BRET, was influenced by cell density.

Thesis (M. Sc.)--University of Canterbury, 2008. / Typescript (photocopy). Includes bibliographical references (p. 143-152). Also available via the World Wide Web.

Thesis (M.S.)--University of North Carolina at Wilmington, 2004. / Includes bibliographical references (leaves : [89]-95).

Thesis (MSc)--University of Stellenbosch, 2008. / Bibliography. Also available via the Internet.

Thesis (doctoral)--Uppsala University, 1983. / Includes bibliographical references (p. 37-48).

No description available.

No description available.

Les travaux de cette thèse ont porté sur le contrôle des neurones à GHRH dans des conditions physiologiques et pathologiques. Le but étant de caractériser les mécanismes cellulaires et moléculaires impliqués dans le fonctionnement ou dérégulations du réseau de neurones à GHRH. Ces neurones sont les principaux stimulateurs de la libération de l’hormone de croissance (GH). Nous avons d’abord montré que l’axe de la croissance et de l’appétit peuvent être régulés indépendamment au niveau de l’hypothamus. En effet, la ghréline, seule hormone produite par le tractus gastro-intestinal et connue pour stimuler la libération de GH en agissant principalement sur les neurones GHRH, stimule ces derniers de manière uniquement directe. Ces effets sont indépendants de ceux qu’elle exerce sur les neurones voisins à NPY, orexigéniques. De plus, la ghréline et les GHS (agonistes sélectifs du récepteur de la ghréline) ne changent pas le mode de décharge électrique des neurones à GHRH ni ne les synchronise. Enfin, ces effets ne présentent pas de dimorphisme sexuel. Dans un second temps, la somatostatine, principal inhibiteur de l’axe GH, induit un rythme d’activité électrique des neurones à GHRH médié par les récepteurs de sous-type SST1 et SST2. Ces effets sont donc temps-dépendants, et aussi sexuellement dimorphiques. Ils sont probablement impliqués dans la modulation de la pulsatilité ultradienne de la libération de GH. Enfin, après un traumatisme crânien, nous observons un déficit de la libération de GH qui apparaît tôt et est soutenu, comme ceux observés chez l’humain. Aucune inflammation ni changement histologique n’a été observe dans l’hypophyse. Cependant, l’inflammation, impliquant une réaction tanycytaire, microgliale, astrocytaire, est présente dans le noyau arqué et l’éminence médiane (EM), ou sont respectivement présents les corps cellulaires et terminaisons des neurones à GHRH. Ceci est lié à des changements morpho-fonctionnels de l’EM (augmentation perméabilité, rupture des barrières tanycytaires). Aucun changement n’a été observé dans le noyau périventriculaire, où sont localisés les neurones à somatostatine. Enfin, les propriétés électriques passives des neurones à GHRH ne sont pas modifiées. En conclusion, une dérégulation de leur activité au niveau des terminaisons nerveuses doit expliquer les défauts posttraumatiques de libération de GH. / The works of this thesis were interested in the control of the hypothalamic GHRH neurons in physiological and pathological conditions. The goal was to clarify the molecular and cellular mechanisms involved in the control or impairments of GHR neuronal network functions. These neurons are the main stimulators of the GH release. We first showed that the hypothalamic growth axis could be regulated independently from the feeding network. Indeed, GHRH neurons are directly stimulated by ghrelin, which is the only hormone produced by the gastrointestinal tract known to stimulate the GH release through acting mainly on GHRH neurons. These effects are independent from its orexigenic effects exerted on the neighbourings NPY neurons. In addition, ghrelin and GHS (synthetic ghrelin receptor agonists) don’t change neither the firing rate of GHRH neurons, nor synchronize them. These effects are not gender-dependant; by contrast, Somatostatin, the major GH axis inhibitor, generates a sexual dimorphic and rhythmic inhibition of the GHRH neurons electrical activity mediated by its SST1 and SST2 receptors subtypes. These effects are so time-dependant direct and indirect effects and can probably be involved in the generation of the ultradian rhythm of the GH release. After a traumatic brain injury, we found an early and sustained deficiency of the GH release, like those observed in human. No pathological changes are visible in the pituitary gland. Inflammation occurs at the arcuate nucleus, and mainly at the median eminence levels; it involves a strong astrocyte reaction, tanycytes, and microglial and (or) infiltrated immune cells activations. These changes elicit morpho-functional impairments of the median eminence, permeability and leakage of the tanycyte barrier between the blood, CSF and Arc; at the opposite, nothing occur at the periventricular level, where are located SST neurons. Neither the number of GHRH neurons, neither their passive electrophysiological properties changed. Impairments of the activities of the GHRH nerve terminals, maybe associated to impairments of their regulated activity, must explain a GH deficiency.

The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on January 6, 2010). Thesis advisor: M. Cathleen Kuehl-Kovarik. Includes bibliographical references.