Dissertations / Theses on the topic 'Gonadotrophin Hormone Releasing Hormone (GnRH)'

Prior studies in the laboratory using quantitative in situ hybridisation and transgenic methodologies have shown that estrogen suppresses GnRH gene transcription and GnRH mRNA expression at times of negative feedback. Using the same approaches, I demonstrated that the positive feedback actions of estrogen were not coupled to alterations of GnRH gene expression. Recent RT-PCR studies identified estrogen receptor (ER)b transcripts in subpopulations of GnRH neurons in the mouse. To assess the presence of functional ERs in GnRH neurons, I undertook various gonadectomy-steroid replacement paradigms in several new transgenic reporter mouse line in which multiple estrogen response elements (EREs) drive the expression of LacZ (EREZ mice). Others have shown that this construct provides a functional reporter of ER-regulated gene transcription in vitro and I demonstrated its functionality in the uterus of EREZ mice. However, LacZ expression in the brain did not change in response to estrogen treatment and did not, therefore, enable us to evaluate the GnRH neurons. One possible reason for this failure is the recent discovery that the orphan nuclear receptors, termed estrogen receptor-related receptors (ERRs), constitutively transactivate EREs. To evaluate this further, I used in situ hybridisation to examine the topography of ERRa and ERRg expression in the mouse brain. I further demonstrated an estradiol-dependent decrease in ERRg expression in specific brain regions, suggesting a novel mechanism through which estradiol may regulate gene expression in the brain. Finally, I examined the negative feedback actions of estrogen upon GnRH neurons by evaluating LH levels and GnRH mRNA expression in knockout mice lacking either ERa or ERb.

Gonadotrophin-releasing hormone (GnRH) can inhibit proliferation of multiple reproductive tissue cancer cell lines through direct interaction with GnRH receptors (GnRHR) on tumour cells. GnRH analogues may therefore have a role in treating some cancers. The signalling pathways associated with these inhibitory effects are poorly defined, and characterising them may help to understand therapeutic sensitivity. To elucidate these pathways, transcriptomic and proteomic approaches were used to compare the effects of the GnRH agonist Triptorelin in responsive GnRHR-transfected HEK293 cells (SCL60) and unresponsive (HEK293) cells both in vitro for up to 24h and in vivo for up to 7 days. Gene expression profiling demonstrated that SCL60 gene expression was temporally regulated with Triptorelin treatment, with expression of some genes increased at one time point but decreased at another. Early and mid-phase gene expression changes comprised mainly transcription factors and late changes included the hormonal signalling component CGA. Pathway analysis implicated mitogen-activated protein kinase and cell cycle pathways, supporting the detection of G2/M arrest. Signalling effects within SCL60 xenografts, 4 and 7 days following Triptorelin treatment, were investigated using a phosphoproteomic antibody array. Changes included cell cycle and apoptosis regulators, as well as cell surface receptors and NFκB signalling pathway members. Reverse-phase protein arrays and western blotting also showed that pAkt was decreased and pNFκB-p65 was increased after Triptorelin treatment in vitro. An NFκB inhibitor enhanced the anti-proliferative effect of Triptorelin in SCL60 cells in vitro, suggesting that NFκB acts as a survival factor in the response to GnRHR stimulation. A range of GnRHR expression was observed in breast cancer tumours by immunohistochemistry, and on average GnRHR expression was significantly higher in the Triple Negative Phenotype (TNP) subgroup and in grade 3 tumours. A GnRHR-transfected breast cancer cell line, MCF7-h14, was developed. Despite this expressing a similar level of GnRHR to responsive SCL60 cells, MCF7-h14 cells were not inhibited by GnRHR activation, indicating that a high level of GnRHR is insufficient for the antiproliferative effects of Triptorelin.

G protein-coupled receptors (GPCRs) are a large family of trans-membrane receptors that transmit signals from extracellular stimuli to target intracellular signal transduction pathways. The gonadotropin-releasing hormone receptor (GnRH-R) is a GPCR which binds the decapeptide GnRH. In the pituitary gonadotrope, GnRH stimulates gonadotropin (LH and FSH) biosynthesis and secretion to regulate reproduction. GnRH and the GnRH-Rs are also present in many extra-pituitary tissues, although their role at these sites remains largely undetermined. GnRH-Rs are known to recruit a diverse array of signalling pathway mediators in different cell-types. These include; Gq/11-PLCβ-IP3/DAG-Ca2+/PKC signalling, monomeric G-proteins and integrins to mediate cell adhesion and migration, the activation of the major members of the mitogen-activated protein kinase (MAPK) super-family (extracellular signal-regulated kinase (ERK), c-Jun N-terminal Kinase (JNK) and p38MAPK), and β-catenin and other mediators of the canonical Wnt signalling pathway. This thesis describes the regulation of Forkhead Box O (FOXO) transcription factors by GnRH. The mammalian FOXO transcription factors, FOXO1, FOXO3a and FOXO4, are emerging as an important family of proteins that modulate the expression of genes involved in cell-cycle regulation, induction of apoptosis, DNA damage repair and response to oxidative stress. In this thesis, emphasis is placed on delineating the novel role of FOXO transcription factors in mediating two important and widely-researched areas of GnRH biology. Firstly, the role of FOXO transcription factors in mediating cell-growth inhibition in response to GnRH treatment is assessed in a heterologous HEK293/GnRH-R expressing cell line. Secondly, the role of transcription factors in regulating luteinising hormone-β (LHβ)-subunit expression is investigated in the LβT2 gonadotrope cell line. Activation of the GnRH-R can inhibit cell proliferation and induce apoptosis in certain tumour-derived cell lines. Several studies have reported that these events can occur as a result of changes in the expression profiles of specific cell-cycle regulatory and apoptotic genes, many of which are FOXO-target genes, including GADD45, FasL, p21Cip1 and p27Kip1. In this thesis, a role for FOXOs in targeting the expression of several of these genes in response to GnRH is assessed, highlighting a specific role for FOXO3a in mediating GADD45 and FasL expression. The signalling mechanisms through which FOXO3a regulates GADD45 expression in response to GnRH is also described. Finally, a stable FOXO3a-knock-down cell line was generated in order to further examine FOXO3a involvement in GnRH-induced cell-growth inhibition. GnRH is an essential regulator of the reproductive process by stimulating the synthesis of LH and FSH in pituitary gonadotropes, thereby regulating gametogenesis and steroidogenesis. Diverse signalling pathways have been reported to regulate LHβ-subunit expression in response to GnRH, including the ERK/JNK/p38MAPK cascades and factors such as Egr1, SF1 and β-catenin. In the second part of this thesis, the role of FOXOs in regulating LHβ-subunit expression in response to GnRH is described. The data presented suggests that GnRH can regulate LHβ-subunit expression through both indirect and direct FOXO3a-mediated mechanisms. Firstly, FOXO3a was found to regulate Egr1 expression to indirectly target LHβ-promoter activity. Secondly, a role for β-catenin as a FOXO3a co-factor to directly regulate LHβ-subunit expression, together with Egr1 and SF1, is also proposed. FOXO3a expression and sub-cellular localisation was assessed and demonstrated in LβT2 cells and in adult human male pituitary sections. The research presented in this thesis adds to the diversity of signalling pathways and mediators that GnRH can target in different cellular backgrounds in order to mediate a variety of cellular processes. The antiproliferative and apoptotic effects of GnRH on tumour-derived cell lines are well-documented, and this research highlights a novel role for FOXO3a in mediating these events. The regulation of gonadotropin synthesis remains an important topic of research, and the novel implication of FOXO3a in mediating LHβ-subunit expression adds further complexity to gonadotrope physiology.

Gonadotropin-releasing hormone (GnRH) and its superactive analogues are currently being used in the treatment of a number of endocrine disorders, such as endometriosis, precocious puberty, infertility and prostatic cancer. Selection of these analogues for clinical use have been previously based on their activities in animal models. This thesis has therefore investigated the binding characteristics of the human GnRH receptor, in comparison to those of the rat receptor, as well as the activities of a number of GnRH analogues for stimulating luteinising hormone (LH) and follicle stimulating hormone (FSH) secretion from cultured human pituitary cells. The establishment of a human pituitary bioassay system has further made possible the investigation of the direct regulatory roles of GnRH and other neuropeptides in man. To date, such studies in man have been performed in vivo and are thus complicated by the simultaneous interactions of numerous modulators.

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
O hormônio liberador de gonadotropina (GnRH) é um dos fatores chaves na regulação neuroendócrina da reprodução dos vertebrados. Alguns peixes apresentam três variantes do GnRH: o GnRH1 envolvido na secreção de gonadotropinas, o GnRH2 que regula o comportamento alimentar e sexual e o GnRH3 expresso no bulbo olfatório e o nervo terminal cujas fibras nervosas inervam a retina e o epitélio olfatório. O zebrafish possui duas variantes do GnRH (GnRH2 e GnRH3), sendo o GnRH3 a variante hipofisiotrófica. Estudos mostram possível envolvimento do GnRH no sistema olfato-retinal. No sistema olfatório o GnRH regula a sensibilidade na detecção de alimento, o reconhecimento intra e interespecífico, entre outros. Na retina, o GnRH3 pode estar envolvido na acuidade visual e do processamento de informação da retina. Existem estudos que reportam a presença de receptores de GnRH em diferentes camadas da retina, no entanto ainda não é clara a presença de receptores no epitélio olfatório. Neste contexto, no presente estudo analisamos a localização do gnrh2, gnrh3 e seus receptores (gnrhr1,2,3 e 4) e do gnih (hormônio inibidor de gonadotropinas) no epitélio olfatório, a retina e o bulbo olfatório de machos e fêmeas adultos e comparamos a expressão destes genes em fêmeas em diferentes estágios de maturação gonadal. Para tanto, o RNA total do epitélio olfatório, retina, bulbo olfatório, cérebro e gônadas foi extraído. Com base na sequência dos genes gnrh2, gnrh3, gnrhr1, gnrhr2, gnrhr3 e gnrhr4, primers forward e reverse foram desenhados para RT-PCR e qPCR. Sondas para a hibridização in situ também foram construídas para verificar os sítios de expressão destas moléculas no epitélio olfatório, retina e gônadas. Imunohistoquímica com os anticorpos anti-GnRH3 (BB8 e GF6) foram realizadas para localizar a proteína do GnRH3 nos tecidos analisados. O presente estudo apresenta um panorama da expressão do sistema...
The gonadotropin releasing hormone (GnRH) is one of the key factors involved in the neuroendocrine regulation of vertebrate reproduction. Some fish species have three GnRH variants: GnRH1 involved in gonadotropin secretion, GnRH2 regulating food and sexual behaviors and the GnRH3 which is expressed in the olfactory bulb and terminal nerve whose fibers innervate the retina and the olfactory epithelium. Two GnRH variants (GnRH2 and GnRH3) are present in the zebrafish, in which GnRH3 acts as the hypophisiotrophic variant. Recent studies have been showing the role of GnRH in the olfactory-retinal system. In the olfactory system, GnRH regulates food detection, and intra and interspecific recognition. In retina, GnRH3 may be involved in visual acuity modulation and retinal processing information. Moreover, studies have reported the presence of GnRH receptors in the retina, but not yet in the zebrafish olfactory epithelium. Therefore, the current study analyzed the presence of GnRH2, GnRH3 and its receptors (GnRH-R1,2,3 and 4) and GnIH (gonadotropin inhibitory hormone) in the olfactory epithelium, olfactory bulb, retina and in gonads of adult zebrafish. We also compared the expression of these genes during the different stages of ovarian maturation in zebrafish. For that, total RNA of the olfactory epithelium, olfactory bulb, retina and gonads was extracted with the PureLink® RNA Mini Kit(Ambion®). RT-PCR and qPCR analysis were performed using forward and reverse primers for gnrh2, gnrh3, gnrhr1, gnrhr2, gnrhr3, gnrhr4 for . Probes for in situ hybridization were constructed to verify the expression sites of these molecules in the olfactory epithelium, retina, and gonads. Immunohistochemistry usinganti-GnRH3 antibodies (BB8 and GF6) were performed to identify the GnRH3 protein in these tissues. The current study presents a general expression view of GnRH/GnIH and their receptors in the olfactory epithelium-olfactory bulb-retinal axis during ...
FAPESP: 2014/02481-9

Orientador: Laura Satiko Okada Nakaghi
Coorientador: Rafael Henrique Nóbrega
Banca: Elisabeth Criscuolo Urbinati
Banca: Matias Pandolfi
Resumo: O hormônio liberador de gonadotropina (GnRH) é um dos fatores chaves na regulação neuroendócrina da reprodução dos vertebrados. Alguns peixes apresentam três variantes do GnRH: o GnRH1 envolvido na secreção de gonadotropinas, o GnRH2 que regula o comportamento alimentar e sexual e o GnRH3 expresso no bulbo olfatório e o nervo terminal cujas fibras nervosas inervam a retina e o epitélio olfatório. O zebrafish possui duas variantes do GnRH (GnRH2 e GnRH3), sendo o GnRH3 a variante hipofisiotrófica. Estudos mostram possível envolvimento do GnRH no sistema olfato-retinal. No sistema olfatório o GnRH regula a sensibilidade na detecção de alimento, o reconhecimento intra e interespecífico, entre outros. Na retina, o GnRH3 pode estar envolvido na acuidade visual e do processamento de informação da retina. Existem estudos que reportam a presença de receptores de GnRH em diferentes camadas da retina, no entanto ainda não é clara a presença de receptores no epitélio olfatório. Neste contexto, no presente estudo analisamos a localização do gnrh2, gnrh3 e seus receptores (gnrhr1,2,3 e 4) e do gnih (hormônio inibidor de gonadotropinas) no epitélio olfatório, a retina e o bulbo olfatório de machos e fêmeas adultos e comparamos a expressão destes genes em fêmeas em diferentes estágios de maturação gonadal. Para tanto, o RNA total do epitélio olfatório, retina, bulbo olfatório, cérebro e gônadas foi extraído. Com base na sequência dos genes gnrh2, gnrh3, gnrhr1, gnrhr2, gnrhr3 e gnrhr4, primers forward e reverse foram desenhados para RT-PCR e qPCR. Sondas para a hibridização in situ também foram construídas para verificar os sítios de expressão destas moléculas no epitélio olfatório, retina e gônadas. Imunohistoquímica com os anticorpos anti-GnRH3 (BB8 e GF6) foram realizadas para localizar a proteína do GnRH3 nos tecidos analisados. O presente estudo apresenta um panorama da expressão do sistema...
Abstract: The gonadotropin releasing hormone (GnRH) is one of the key factors involved in the neuroendocrine regulation of vertebrate reproduction. Some fish species have three GnRH variants: GnRH1 involved in gonadotropin secretion, GnRH2 regulating food and sexual behaviors and the GnRH3 which is expressed in the olfactory bulb and terminal nerve whose fibers innervate the retina and the olfactory epithelium. Two GnRH variants (GnRH2 and GnRH3) are present in the zebrafish, in which GnRH3 acts as the hypophisiotrophic variant. Recent studies have been showing the role of GnRH in the olfactory-retinal system. In the olfactory system, GnRH regulates food detection, and intra and interspecific recognition. In retina, GnRH3 may be involved in visual acuity modulation and retinal processing information. Moreover, studies have reported the presence of GnRH receptors in the retina, but not yet in the zebrafish olfactory epithelium. Therefore, the current study analyzed the presence of GnRH2, GnRH3 and its receptors (GnRH-R1,2,3 and 4) and GnIH (gonadotropin inhibitory hormone) in the olfactory epithelium, olfactory bulb, retina and in gonads of adult zebrafish. We also compared the expression of these genes during the different stages of ovarian maturation in zebrafish. For that, total RNA of the olfactory epithelium, olfactory bulb, retina and gonads was extracted with the PureLink® RNA Mini Kit(Ambion®). RT-PCR and qPCR analysis were performed using forward and reverse primers for gnrh2, gnrh3, gnrhr1, gnrhr2, gnrhr3, gnrhr4 for . Probes for in situ hybridization were constructed to verify the expression sites of these molecules in the olfactory epithelium, retina, and gonads. Immunohistochemistry usinganti-GnRH3 antibodies (BB8 and GF6) were performed to identify the GnRH3 protein in these tissues. The current study presents a general expression view of GnRH/GnIH and their receptors in the olfactory epithelium-olfactory bulb-retinal axis during ...
Mestre

La gonadotropin releasing hormone est essentielle à la reproduction. Sécrétée de manière pulsatile, elle contrôle la sécrétion des hormones gonadotropiques hypophysaires. Les neurones à GnRH, peu nombreux, dispersés dans l'aire préoptique, ont une origine extracérébrale. Une phase migratoire au cours de la vie embryonnaire précède la mise en place de la sécrétion. Les mécanismes de la migration et de la sécrétion, en particulier la pulsatilité, restent méconnus. Avec des cultures d'explants olfactifs, nous avons étudié les conductances calciques au cours du développement et les facteurs impliqués dans la synchronisation. Nos résultats montrent une maturation des conductances calciques et leur contribution relative dans les fluctuations calciques intracellulaires au cours du développement. Quant à la synchronisation, nos résultats montrent l'existence d'une rythmicité dans l'activité électrique des neurones et l'implication du GABA et de la GnRH sans écarter d'autres neurotransmetteurs
The gonadotropin releasing hormone is essential to the reproduction. Secreted in a pulsatile manner, it controls the gonadotropic hormones secretion. GnRH neurons are in small number and are spread in the hypothalamic preoptic area. A migratory phase during the embryonic life, precedes the set up of the secretion. Mechanisms involved in the migratory state, as well as those involved in secretion, in particularly pulsatility, remain unknown. Using two models of nasal explants cultures, we studied modifications in calcium conductances during the in vitro development and factors potentially involved in neuronal synchronization. Our results have underlined a maturation in calcium conductances and their relative contribution in intracellular calcium signaling occurring during the in vitro development. They have also shown the existence of rhythmicity in electrical activity, and confirmed the role for GABA and GnRH, even if the involvement of others neurotransmitters cannot be eliminated

Thesis (MSc)--Stellenbosch University, 2001.
ENGLISH ABSTRACT: The GnRH receptor is a G-protein-coupled receptor in pituitary gonadotrope cells. Binding of its ligand, GnRH, results in synthesis and release of gonadotropin hormones luteinizing hormone (LH) and follicle stimulating hormone (FSH). Steroidogenic factor 1 (SF-1), a transcription factor, binds to specific sites in the promoter region of gonadotropin genes, and thus regulates transcription of these genes. The promoter region of the GnRHreceptor gene contains two SF-1-like binding sites, one at -14 to -8 (site 1) and another at -247 to -239 (site 2), relative to the methionine start codon. The role played by these two SF-1-like sites in basal transcription of the mouse GnRH receptor (mGnRH-R) gene in a pituitary precursor gonadotrope cell line, aT3 cells, was the first area of investigation during this study. Luciferase reporter constructs containing 580 bp of mGnRH-R gene promoter were prepared, where SF-1-like sites were either wildtype or mutated. Four such constructs were made, i.e. wildtype (LG), site 1 mutant (LGM1), site 2 mutant (LGM2) and mutated site 1 plus site 2 (LGM1/2). These constructs were transfected into aT3 cells to determine the effect of mutations of sites 1 and/or 2 on the basal expression of the mGnRH-R gene. Mutation of either site 1 or site 2 had no effect on basal expression of the mGnRH-R gene. It was found that only upon simultaneous mutation of both sites 1 and 2, a 50% reduction in basal transcription took place. The implications of this is that SF-1 protein seems to only require one intact DNA-binding site, to mediate basal transcription of the mGnRH-R gene, suggesting that these two sites lie in close proximity during basal transcription. The effect of the protein kinase A (PKA) pathway on the endogenous mGnRH-R gene was also investigated by incubating non- , transfected aT3 cells with the PKA activators, forskolin and 8-Br-cAMP. Similar incubations were also performed on the wild type and mutated site 1 constructs transfected into pituitary gonadotrope aT3 cells. It was found that forskolin and 8-Br-cAMP were able to increase endogenous mGnRH-R mRNA levels in a concentration-dependent fashion, showing that endogenous GnRH receptor gene expression is stimulated via a protein kinase A pathway. Similar results were obtained with the wildtype promoter construct, showing that the protein kinase A pathway stimulates transcription of the promoter. This effect was only seen with wild type and not with the mutated site 1. These results are consistent with a role for a SF-1-like transcription factor in mediating the protein kinase A effect via binding to the site 1 at position -14 in the GnRH receptor gene. A separate investigation was performed to determine whether 25-hydroxycholesterol (25-0HC) is a ligand for SF-1, by incubating aT3 cells transfected with the various constructs with 25-0HC. Results show a dose-dependant response, with an increase in gene expression at 1 μM and a decrease at higher concentrations, for both mutant and wild type constructs. This suggests that, if SF-1 is indeed the protein binding to sites 1 and 2, then 25-0HC is not a ligand for SF-1 protein in aT3 cells and that the effect of 25-0HC on the mGnRH-R gene is not mediated via site 1. The results indicate that these decreases of expression at the higher concentrations may be due to cytotoxic effects. Towards the end of the study the laboratory obtained a luminoskan instrument with automatic dispensing features. Optimisation studies on the luciferase and β-Gal assays were performed on the luminoskan in a bid to decrease experimental error. It was found that automation of these assays resulted in a decrease in experimental error, showing that future researchers could benefit substantially from these optimisation studies.
AFRIKAANSE OPSOMMING: Die GnRH reseptor is 'n G proteïen-gekoppelde reseptor in pituitêre gonadotroopselle. Binding van die ligand, GnRH, lei tot die sintese en vrystelling van die gonadotropien hormone, luteïniserende hormoon (LH) en follikel stimulerende hormoon (FSH). Steroidogeniese faktor-t (SF-1) is 'n transkripsie faktor wat aan spesifieke areas in die promotergebied van die gonadotropien hormone bind, en dus transkripsie van hierdie gene reguleer. Die promotergebied van die GnRH reseptor geen bevat twee SF-1 bindings areas, een by -14 to -8 (area 1) asook by -247 to -239 (area 2), relatief to die metionien beginkodon. Die rol wat hierdie twee SF-1 areas speel in basale transkripsie van die muis GnRH reseptor (mGnRH-R) geen in 'n pituïtêre voorloper gonadotroop sellyn, aT3 selle, was die eerste gebied van ondersoek gedurende hierdie studie. Plasmiede bestaande uit die 580 basispaar mGnRH-R promoter verbind aan 'n lusiferase geen is vervaardig, waar SF-1-soortige areas enersyds onveranderd gelaat is, of gemuteer is. Vier sulke plasmiede is vervaardig, nl. onveranderd (LG), area 1 mutant (LGM1), area 2 mutant (LGM2) en gemuteerde area 1 plus area 2 (LGM1/2). Hierdie plasmiede is gebruik om aT3 selle te transfekteer om die effek van mutasies van areas 1 en/of 2 op die basale ekspressie van die mGnRH-R geen te ondersoek. Daar is gevind dat mutasies van areas 1 of 2 geen effek op basale ekspressie op die bogenoemde geen gehad het nie. Slegs tydens gelyktydige mutasie van areas 1 en 2 het 'n 50% vermindering in basale transkripsie plaasgevind. Die implikasies hiervan is dat die SF-1 proteïen blykbaar slegs een volledige DNA-bindingsarea benodig om basale transkripsie van die mGnRH-R geen te reguleer. Dit wil dus voorkom of hierdie twee areas baie na aan mekaar geposisioneer is tydens basale transkripsie. Die effek van die proteïen kinase A (PKA) roete op die natuurlike mGnRH-R geen is ook ondersoek tydens inkubasie van nie-getransfekteerde aT3 selle met die PKA akiveerders, forskolin en 8-Br-cAMP. Soortgelyke inkubasie is ook gedoen op die onveranderde en gemuteerde area 1 plasmiede wat in aT3 selle getransfekteer is. Daar is gevind dat forskolin en 8-Br-cAMP daarin geslaag het om die natuurlike mGnRH-R geen mRNA vlakke op 'n konsentrasie-afhanklike wyse te vermeerder. Hierdie resultaat dui daarop aan dat die natuurlike mGnRH-R geen se ekspressie gestimuleer kan word via 'n proteïen kinase A roete. Soortgelyke resultate is verkry met die onveranderde promoter plasmied en dit wys ook daarop dat proteïen kinase A transkripsie deur die promoter kan stimuleer. Hierdie effek was slegs aanwesig met die onveranderde en nie met die gemuteerde area 1 plasmied nie. Die resultate stem ooreen met 'n rol vir SF-1 transkripsie faktor in die regulering van proteren kinase A effek deur middel van binding aan die area 1 by posisie -14 in die GnRH-R geen. 'n Afsonderlike ondersoek is gedoen om vas te stel of 25-hidroksiecholesterol (25-0HC) 'n ligand vir SF-1 is deur getransfekteerde aT3 selle met 25-0HC te inkubeer. Resultate toon 'n dosis-afhanklike respons met 'n verhoging in geen ekspressie by 1 μM en 'n verlaging met hoër konsentrasies vir beide onveranderde en gemuteerde plasmiede. Dit impliseer dat, indien SF-1 wel die faktor is wat aan areas 1 en 2 bind, 25-0HC nie die ligand vir SF-1 proteren in aT3 selle is nie en dat die effek van 25-0HC op die mGnRH-R geen nie gereguleer word via area 1 nie. Die verlaging in ekspressie gevind by die hoër konsentrasies is dalk die gevolg van sitotoksiese effekte. Teen die einde van die studie het die laboratorium luminoskan toerusting met outomatiese pipettering verkry. Optimiseringstudies van die lusifirase en β-Galtoetse is met die luminoskan gedoen in 'n poging om eksperimentele foute te minimaliseer. Daar is gevind dat outomatisering van hierdie toetse wel gelei het tot 'n verlaging in eksperimentele foute. Toekomstige navorsers kan dus grootliks voordeel trek uit hierdie optimiseringstudies.

[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.

Objectives were to determine if low-dose, continuous infusion of GnRH from Fall to Spring, would prevent seasonal anovulation in mares. Twenty Quarter Horse mares, ages 18 mo to 24 yrs, were stratified by age and body condition score and assigned randomly to either a saline control (n = 9) or GnRH (n = 11) treatment group. Treatments were instituted between September 23 and October 9, 2002. Gonadotropinreleasing hormone was delivered in 0.9% physiological saline via Alzet osmotic minipumps (Model 2004) placed sc at the base of the neck, with Silastic sham pumps placed in control mares. Pumps were inserted on day 3 following ovulation or during the follicular phase if ovulation had not occurred. Delivery rate of GnRH was 2.5 ug/h (60 ug/d) for the first 60 d, followed by 5.0 ug/h (120 ug/d) thereafter, with all pumps replaced every 30 d. By December 1, all mares had become anovulatory and remained anovulatory until February. Mean serum concentrations of LH were not affected by treatment in anovulatory mares. In contrast, control mares that exhibited ovulatory cycles after treatment onset had higher (P < 0.05) mean concentrations of LH during all phases of the estrous cycle except diestrus. Mean serum concentrations of FSH were not affected by treatment, but were lower (P < 0.05) from November though January relative to all other months in anovulatory mares. Interovulatory intervals in mares that cycled temporarily did not differ between groups. Ovulatory control mares had slightly larger (P < 0.10) follicles overall than GnRH-treated mares; however, ovulatory follicle diameters for control and GnRH-treated mares did not differ. Ovulatory control mares had higher (P < 0.10) mean concentrations of progesterone during metestrus and late diestrus. In a subgroup of control (n =5) and GnRH-treated (n = 5) mares, total releasable pools of LH in response to 1 mg GnRH did not differ between groups. Ovulation resumed in 3 control and 3 GnRH-treated mares by March 30. Results indicate that continuous infusion of native GnRH at the doses employed herein is not sufficient to maintain ovulatory cycles during the anovulatory season.

Includes abstract.
Includes bibliographical references.
Includes bibliographical references (leaves 74-81).
Over-expression of β-arrestin 1 in COS-l cells revealed that the mammalian type GnRH receptor can internalise in a β-arrestin dependent manner whereas the internalisation of the mammalian type I GnRH receptor is β-arrestin independent. investigate which domains on the mammalian type II GnRH receptor are required for β~arrestin dependent internalisation, chimeric receptors were created.
The mammalian type II GnRH receptor possesses an intracellular C-terminal tail that is known to play a role in desensitisation, internalisation and overall signalling in GPCRs. On the other hand, the mammalian type I GnRH receptor, which lacks a C-terminal tail, does not readily desensitise and undergoes slow internalisation compared to the mammalian type II GnRH receptor. Over-expression of ß-arrestin 1 in COS-l cells revealed that the mammalian type GnRH receptor can internalise in a ß-arrestin dependent manner whereas the internalisation of the mammalian type I GnRH receptor is ß-arrestin independent. investigate which domains on the mammalian type II GnRH receptor are required for ß-arrestin dependent internalisation, chimeric receptors were created. Firstly, a chimera in which the full length type II GnRH receptor C-terminal tail was added to the tail-less type I GnRH receptor (TI/T2tail) was created. This chimera internalised in a ß-arrestin and GRK dependent manner, demonstrating that the type II GnRH receptor C-terminal tail confers ß-arrestin JGRK dependent internalisation on the originally ß-arrestin/GRK insensitive GnRH receptor. Mutating the putative GRK and casein kinase phosphorylation sites (serines 338 and 339) on the C-terminal tail of TI/T2tail to alanine residues did not abolish ß-arrestin dependent internalisation but eliminated GRK dependent internalisation, suggesting that other regions on the C-terminal tail are required for ß-arrestin dependent internalisation. A second chimera, in which the whole third intracellular loop of the type II GnRH receptor was replaced with that of the type I GnRH receptor (T2/TIICL3), was created. This chimera could not utilise ß-arrestin in its internalisation, indicating that the third intracellular loop of the type II GnRH receptor is required for ß-arrestin dependent internalisation. An alignment of the amino acid sequences of the two mammalian GnRH receptor third intracellular loops identified a basic residue rich area (R234, R236 and K237) on the type II GnRH receptor that was absent on the type I GnRH receptor. Interestingly, the triple mutant (R234,236,K237 A) still internalised in a ß-arrestin dependent manner, however, truncation of the C-terminal tail of R234,236,K237A abolished the ability of the receptor to internalise in a ß-arrestin dependent manner. This result indicated that the C-terminal tail of the type II GnRH receptor was compensating for the absence of the three basic residues. To summarise, this thesis demonstrates that the C-terminal tail of the type II GnRH receptor can confer ß-arrestin dependent intemalisation on the type I GnRH receptor. Furthermore, the third intracellular loop, and more specifically, basic residues R234, R236 and K237 on the mammalian type II GnRH receptor are required for ß-arrestin dependent intemalisation.

The plasma membrane electrical activities of neurons that secrete gonadotropin releasing hormone (GnRH), referred to as GnRH neurons hereafter, have been studied extensively. A couple of mathematical models have been developed previously to explain different aspects of these activities including spontaneous spiking and responses to stimuli such as current injections, GnRH, thapsigargin (Tg) and apamin. The goal of this paper is to develop one single, minimal model that accounts for the experimental results reproduced by previously existing models and results that were not accounted for by these models. The latter includes two types of membrane potential bursting mechanisms and the associated calcium oscillations in the cytosol. One of them has not been reported in experimental literatures on GnRH neurons and is thus regarded as a model prediction. Other improvements achieved in this model include the incorporation of a more detailed description of calcium dynamics in a three dimensional cell body with the ion channels evenly distributed on the cell surface. Although the model is mainly based on data collected in cultured GnRH cell lines, we show that it is capable of explaining some properties of GnRH neurons observed in several of other preparations including mature GnRH neurons in hypothalamic slices. One potential explanation is suggested. A phenomenological reduction of this model into a simplified form is presented. The simplified model will facilitate the study of the roles of plasma membrane electrical activities on the pulsatile release of GnRH by these neurons when it is coupled with a model of pulsatile GnRH release based on the autoregulation mechanism.

Two experiments were conducted to determine efficacy of GnRH analogs, Cystorelin (CYS, gonadorelin diacetate tytrahydrate) and Factrel (FAC, gonadorelin hydrochloride), for use in beef timed AI synchronization. In Experiment one 342 beef cows from 7 herds were assigned CYS or FAC treatment as part of the Ovsynch protocol (GnRH d 0 and 9, Lutalyse d 7). Cattle treated with FAC had greater tendency (P=.09) to be pregnant at d 45. One individual herd demonstrated FAC-treated cows had more pregnancies at day 45. In Experiment two, 18 beef cows received either CYS or FAC as part of the Ovsynch protocol, intensive blood samples, from time -30 to 525 min post GnRH, were collected at each GnRH injection. Ultrasounds were conducted daily over the course of the protocol. A treatment by phase interaction (P=.03) was found for the time to maximum LH concentration, where CYS-treated follicular cows had a shorter interval than did FAC treated follicular or luteal cows. The duration of detectable LH response showed a treatment by phase interaction (P = .02) where follicular and luteal CYS-treated cows had shorter interval than follicular or luteal FAC-treated cows. The variables maximum LH concentration, and area under LH curve did not differ. Cows treated with CYS had more (P=.02) non-dominant follicles. In Experiment three, 16 ewes randomly received either CYS, FAT or Fertagyl (FER; gonadorelin diacetaate tytrahydrate), and FAT's induced LH maximum concentration occurred sooner (P=.02) than CYS. We conclude that either product may be used in beef cows without compromising fertility.
Master of Science

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Fundação para o Desenvolvimento da UNESP (FUNDUNESP)
As alterações nos componentes reprodutivos do eixo hipotálamo-hipófise-gônadas em muitas fêmeas de mamíferos determinam a transição gradual de ciclos reprodutivos regulares para ciclos irregulares, com perda de fertilidade. A interação dos neurônios do hormônio liberador de gonadotrofinas (GnRH) e esteróides gonadais representa função chave na neurobiologia do envelhecimento, pois a sobreposição temporal da senescência endócrina e neural está mecanicamente interligada pelas alças de retroalimentação. Estímulos do locus coeruleus (LC) para a área pré-óptica (APO) e eminência mediana são essenciais para a liberação das gonadotrofinas e seus neurônios apresentam receptores para estrógeno e progesterona, sugerindo controle dos esteróides ovarianos. Neste estudo foi avaliado a atividade de células neuronais localizadas em áreas e núcleos envolvidos com o controle de ação dos neurônios GnRH de ratas Wistar no período de transição para a aciclicidade. Para este trabalho foram utilizadas fêmeas Wistar cíclicas (4 meses) e acíclicas (18-20 meses) submetidas à decapitação ou perfusão às 10, 14 e 18 h na fase do diestro. Após serem retirados, os cérebros dos animais decapitados foram congelados e armazenados para posterior determinação do conteúdo de GnRH hipotalâmico e do conteúdo de noradrenalina e dopamina na APO. Os cérebros perfundidos foram cortados seriadamente em secções coronais de 30 μm para a APO e o LC e...
Changes in reproductive components of the hypothalamic-pituitary-gonadal axis in many female mammals determine the gradual transition from regular reproductive cycles to irregular cycles, with loss of fertility. The interaction of neurons of gonadotropin-releasing hormone (GnRH) and gonadal steroids represents key role in the neurobiology of aging, because the temporal overlap of endocrine and neural senescence is mechanically interconnected by feedback loops. Stimulation of the locus coeruleus (LC) for the preoptic area (POA) and median eminence are essential for the release of gonadotropins and their neurons have receptors for estrogen and progesterone, suggesting control of ovarian steroids. Therefore, in this study we evaluated the activity of neuronal cells located in areas and nuclei involved in the control of action of GnRH neurons of female rats during the transition to acyclicity. For this study, we used cyclic female (4 months) and acyclic (18-20 months) rats underwent perfusion or decapitation at 10, 14 and 18 h of diestrus day. The brains from decapitated animals, after removed, were frozen and stored for subsequent determination of the hypothalamic GnRH content and the noradrenaline and dopamine content in the POA. The perfused brains were serially cut into coronal sections of 30 μm to POA and LC and subsequently submitted to immunohistochemical labeling for Fos (FRA) and FRA / TH, respectively. For quantitative analysis of the POA were considered plates containing AVPe being the counting of neurons FRA-ir performed from the insertion of the box with...
FAPESP: 12/14464-6

Orientador: Rita Cássia Menegati Dornelles
Co-orientador: Janete Aparecida Anselmo-Franci
Banca: Maristela de Oliveira Poletini
Banca: Jacqueline Nelisis Zanoni
Resumo: As alterações nos componentes reprodutivos do eixo hipotálamo-hipófise-gônadas em muitas fêmeas de mamíferos determinam a transição gradual de ciclos reprodutivos regulares para ciclos irregulares, com perda de fertilidade. A interação dos neurônios do hormônio liberador de gonadotrofinas (GnRH) e esteróides gonadais representa função chave na neurobiologia do envelhecimento, pois a sobreposição temporal da senescência endócrina e neural está mecanicamente interligada pelas alças de retroalimentação. Estímulos do locus coeruleus (LC) para a área pré-óptica (APO) e eminência mediana são essenciais para a liberação das gonadotrofinas e seus neurônios apresentam receptores para estrógeno e progesterona, sugerindo controle dos esteróides ovarianos. Neste estudo foi avaliado a atividade de células neuronais localizadas em áreas e núcleos envolvidos com o controle de ação dos neurônios GnRH de ratas Wistar no período de transição para a aciclicidade. Para este trabalho foram utilizadas fêmeas Wistar cíclicas (4 meses) e acíclicas (18-20 meses) submetidas à decapitação ou perfusão às 10, 14 e 18 h na fase do diestro. Após serem retirados, os cérebros dos animais decapitados foram congelados e armazenados para posterior determinação do conteúdo de GnRH hipotalâmico e do conteúdo de noradrenalina e dopamina na APO. Os cérebros perfundidos foram cortados seriadamente em secções coronais de 30 μm para a APO e o LC e...
Abstract: Changes in reproductive components of the hypothalamic-pituitary-gonadal axis in many female mammals determine the gradual transition from regular reproductive cycles to irregular cycles, with loss of fertility. The interaction of neurons of gonadotropin-releasing hormone (GnRH) and gonadal steroids represents key role in the neurobiology of aging, because the temporal overlap of endocrine and neural senescence is mechanically interconnected by feedback loops. Stimulation of the locus coeruleus (LC) for the preoptic area (POA) and median eminence are essential for the release of gonadotropins and their neurons have receptors for estrogen and progesterone, suggesting control of ovarian steroids. Therefore, in this study we evaluated the activity of neuronal cells located in areas and nuclei involved in the control of action of GnRH neurons of female rats during the transition to acyclicity. For this study, we used cyclic female (4 months) and acyclic (18-20 months) rats underwent perfusion or decapitation at 10, 14 and 18 h of diestrus day. The brains from decapitated animals, after removed, were frozen and stored for subsequent determination of the hypothalamic GnRH content and the noradrenaline and dopamine content in the POA. The perfused brains were serially cut into coronal sections of 30 μm to POA and LC and subsequently submitted to immunohistochemical labeling for Fos (FRA) and FRA / TH, respectively. For quantitative analysis of the POA were considered plates containing AVPe being the counting of neurons FRA-ir performed from the insertion of the box with...
Mestre

Includes abstract.
Includes bibliographical references (leaves 72-78).
The activation of Gonadotropin-releasing hormone receptor (GnRHR) by the GnRH ligand has been shown to mediate antiproliferative effects in extra-pituitary cells and in reproductive cancer cell lines. The GnRHR couples to Gαq in pituitary gonadotropes. However, the GnRHR expressed in reproductive cancer cell lines is thought to couple to Gαi. Recent evidence also suggests that the antiproliferative effects may be mediated via Gαq in these cells. Therefore our study involved determining the role of Gαi in the antiproliferative effects mediated by the GnRHR. The results suggest that the Gαi pathway could play a role in mediating the antiproliferative effects of GnRH.

Prostaglandin F2a (PGF2a), oxytocin and gonadotropin releasing hormone (GnRH) have been used in bulls, rams, boars, stallions or rodents to increase sperm numbers in the ejaculate. Improving sperm quantity in the canine ejaculate would benefit all assisted reproductive techniques used in this species. The purpose of the present study was to evaluate the effects of PGF2a, oxytocin and GnRH on canine ejaculate characteristics. Eight, mature, medium size (25-30 kg), mixed breed dogs were randomly assigned to one of four treatment groups (N=2 dogs each); each group received one treatment per week for four weeks. Treatments were assigned based on a Latin Square design. A two-week training period was used to acclimate the dogs to manual semen collection. Treatments were 0.1 mg/kg PGF2a 15 minutes prior to collection, 2.5 units/dog oxytocin 10 minutes prior to collection, 50 mg/dog GnRH 60 minutes prior to collection, or 1.0 ml of saline 30 minutes prior to collection. An evaluator that was blinded to treatment analyzed ejaculate characteristics. Samples were evaluated for semen volume, concentration of spermatozoa per milliliter, motility, morphology, total sperm number and total morphologically normal motile sperm number (TNMS). In addition, a subjective ease of collection score was assigned following each collection (Scale 1-9, 1 being easiest to manually ejaculate). Semen concentration, motility and morphology were not different between treatments. Semen volume was greater for dogs treated with PGF2a or oxytocin compared to saline. Total sperm number and TNMS were greater when dogs were treated with PGF2a compared to oxytocin, GnRH and saline (p<0.05). The subjective ease of collection score was lower for dogs receiving PGF2a compared to GnRH or saline (p<0.05). In summary, administration of PGF2a or oxytocin prior to semen collection increased semen volume and PGF2a increased total sperm number in the ejaculate of the dog. It did not appear that treatment with GnRH had an effect on semen parameters evaluated in this study.
Master of Science

Gonadotropin releasing hormone (GnRH) is a key reproductive hormone in vertebrates and exerts its effects via the GnRH receptor (GnRHR) to result in the synthesis and release of the gonadotropin hormones in the pituitary gonadotrope cells. GnRHR expression is likely to be regulated in a tissue- and cell- specific manner. A variety of hormones, including GnRH itself, estrogen, progesterone, inhibin, and testosterone have been shown to regulate GnRHR expression. Steroidogenic Factor-1 (SF-1), a member of the orphan nuclear receptor transcription factor family, regulates the expression of both the gonadotropin hormones in the pituitary and the steroidogenic enzymes in the gonads and adrenal gland, and provides a potential molecular mechanism for coordinate control of reproductive function. SF-1 binds to a gonadotrope-specific element (GSE) in the promoters of the gonadotropin hormones. Our studies involved investigating whether SF- I plays a role in tissue-specific regulation of GnRHR gene expression. A genomic clone of the mouse GnRHR gene contains a putative SF- I site at about -15 relative to the translation start site. We demonstrate the presence of a factor with SF-1-like DNA-binding activity in the gonadotrope cell lines, αT3-1 and αT4, by gel retardation assays. DNasel footprinting reveals that the major DNA-binding activity in αT3-1 cells on the GnRHR promoter occurs at the SF-1-like site. The SF-1-like sequence specificity of the interaction is demonstrated by gel redardation and DNasel footprinting assays using specific and mutated oligonucleotides as competitors. Northern blot analysis suggests that GnRHR expression is not solely dependent on the presence of SF-1, as αT4 cells do not express GnRHR but a SF-1 transcript is seen in these cells. Promoter function was analysed by constructing plasmids containing 563 bp of the GnRHR gene 5' to the ATG translation start site linked to a luciferase reporter gene, followed by transfection of these constructs into different cell lines. In addition, a mutant construct containing a mutated SF-1 site was tested. We demonstrate that this 563 bp of the GnRHR gene contains strong promoter activity in both pituitary gonadotrope (αT3-1) and somatotrope (GH₃) cells, but not in non-pituitary (COS-1) cells. Thus promoter activity appears to be tissue specific but not gonadotrope specific. The presence of a mutated SF-1 site in the 563 bp GnRHR gene fragment did not significantly effect the promoter activity, showing that binding of SF-1 protein to this site is not necessary for high levels of GnRHR expression in the pituitary gonadotropes.

The aim in the present study was to evaluate the impact of treatment with a slow release implant containing the GnRH agonist deslorelin on the pituitary gonadal axis and on sperm production in adult male dogs. The reason for the study was to explore the potential of these implants to achieve long term, reversible contraception in dogs. Three dose rates of deslorelin, 3mg, 6mg and 12mg were used in the implants in the study. The effect of the implants on pituitary and testicular function was monitored by measuring plasma LH and testosterone concentrations, testicular volume and semen characteristics. The degree of desensitization of the pituitary gonadotrophs and Leydig cells during treatment with deslorelin was measured by challenging the dogs with injections of GnRH and bovine LH at 0, 15, 25, 40, and 100 days after implantation, and measuring the plasma LH and testosterone responses to challenge. The effect of the deslorelin implants on the reproductive physiology of the dogs was characterized by a reduction in plasma LH and testosterone concentrations to undetectable levels within 3 weeks of implantation. Histological findings showed atrophic changes in seminiferous tubules, ductus epididymides and prostate gland after 25 days of treatment, the atrophy becoming progressively more severe on day 40 and 100 after implantation. Only spermatogonia and Sertoli cells were present in seminiferous tubules after 100 days.

De nombreux agents chimiothérapeutiques sont gonadotoxiques et peuvent donc induire une insuffisance ovarienne précoce chez les jeunes patientes traitées. La protection pharmacologique de l'ovaire pendant la chimiothérapie à l'aide d'analogues de la Gonadotropin-Releasing Hormone (GnRHa) est une option intéressante de préservation de la fertilité de par son caractère non-invasif et la possibilité d’une récupération spontanée de la fonction ovarienne. Ces molécules sont des inhibiteurs bien connus de l'axe hypothalamo-hypophyso-gonadique, mais leur efficacité dans cette indication est, cependant, controversée et leurs mécanismes d'action sont mal compris. Par conséquent, nous avons investigué les mécanismes potentiels de protection ovarienne des GnRHa pendant la chimiothérapie sur modèle murin. Nous avons montré que le cyclophosphamide (Cy) induit une déplétion folliculaire aiguë et proportionnelle à la dose affectant à la fois les follicules quiescents et en croissance. Lorsqu'ils sont administrés seuls à différentes doses et sites, l'agoniste et l'antagoniste de la GnRH altèrent les cycles oestraux, mais ne bloquent ni la folliculogenèse ni la sécrétion de la Follicle-Stimulating Hormone (FSH) chez la souris. De plus, le Cy atteint les follicules primordiaux, que les souris aient été traitées avec les GnRHa ou non. Ces résultats suggèrent que les GnRHa n'inhibent pas l'axe hypophyso-gonadique aussi efficacement chez la souris que chez la femme. Par conséquent, nous avons développé de nouveaux modèles pour étudier les mécanismes potentiels de protection ovarienne des GnRHa. Afin de différencier les effets directs des GnRHa via leurs récepteurs ovariens ou indirects par inhibition de la sécrétion de gonadotrophines, l'effet de l'agent alkylant sur le développement folliculaire et la réserve ovarienne a été testé sur des follicules cultivés in vitro avec ou sans GnRHa et in vivo chez des souris déficientes en FSHb (Fshb-/-). Pour imiter la profonde inhibition de FSH observée chez la femme après traitement aux GnRHa, nous avons étudié la toxicité de la chimiothérapie chez les souris Fshb-/-. L’administration de gonadotrophines exogènes (pregnant mare serum gonadotropin, PMSG) induit une croissance folliculaire jusqu’au stade antral mais n’influence pas le nombre total de follicules au sein de l’ovaire. Le Cy induit une perte folliculaire significative dans le groupe contrôle et dans le groupe traité au PMSG. Aucune différence concernant la prolifération ni l'apoptose n'a été observée entre les groupes traités à la chimiothérapie. A ce jour, ce modèle murin représente le meilleur modèle pour étudier l'inhibition gonadotrope induite par les GnRHa observée chez la femme. Ces résultats suggèrent que la FSH n'est pas impliquée dans la protection ovarienne potentielle des GnRHa pendant la chimiothérapie. Afin d’évaluer les effets directs des GnRHa sur les follicules en croissance et quiescents, des follicules préantraux ou des ovaires de nouveau-nés (PND4) ont été cultivés avec ou sans GnRHa avant l'exposition au métabolite actif du Cy, le 4-hydroperoxycyclophosphamide (4HC). Nous avons d'abord montré que l'exposition in vitro aux GnRHa n'affectait ni la survie et le développement folliculaire, ni la maturation ovocytaire. Dans les follicules en croissance, le 4HC diminue significativement les taux de survie et de maturation; et retarde le développement folliculaire, indépendamment du traitement aux GnRHa. La chimiothérapie diminue le nombre de cellules de la granulosa par follicule tandis que la production d’adénosine monophosphate cyclique (AMPc) par million de cellules de la granulosa n'est pas modifiée, ni par le 4HC, ni par les GnRHa. La sécrétion d'oestradiol tend à être retardée dans le groupe traité à l’agoniste mais pas dans le groupe antagoniste. De même, dans les ovaires PND4, le 4HC induit une perte folliculaire importante et atteint directement les cellules de la granulosa des follicules ovariens. Aucune différence dans la distribution folliculaire, la prolifération ou l'apoptose n'a été observée entre les groupes traités avec le 4HC, peu importe la présence des GnRHa ou non. Pour conclure, en se basant sur des modèles murins robustes et originaux, notre travail remet en question l'efficacité des GnRHa pour préserver l'ovaire contre les dommages causés par la chimiothérapie que ce soit par une action directe sur l'ovaire, ou indirectement par l'absence de FSH. D'autres investigations seront nécessaires pour comprendre les mécanismes d'action potentiels des GnRHa sur l'ovaire et les voies impliquées. Des preuves expérimentales sont encore indispensables pour clore le débat sur cette option attrayante de préservation de la fertilité.
Doctorat en Sciences biomédicales et pharmaceutiques (Médecine)
info:eu-repo/semantics/nonPublished

Hypothalamic gonadotropin-releasing hormone (GnRH) (GnRH I) is the central regulator of the mammalian reproductive system. Most vertebrates studied also possess a second form of GnRH, GnRH II. GnRH I acts on its cognate G proteincoupled receptor (GPCR) on pituitary gonadotropes and activates Gq/11-mediated signalling pathways to stimulate the biosynthesis and the release of luteinising hormone (LH) and follicle-stimulating hormone (FSH). Both GnRHs have also been suggested to inhibit cellular proliferation, an action which has largely been proposed to be mediated by the coupling of the receptor to Gi/o. However, the range of G proteins activated by the GnRH receptor and the signalling cascades involved in inducing antiproliferation remain controversial. To delineate the G protein coupling selectivity of the mammalian GnRH receptor and to identify the signalling pathways involved in GnRH I-mediated cell growth inhibition, I examined the ability of the receptor to interact with Gq/11, Gi/o and Gs in Gαq/11 knockout MEF cells. My results indicate that the receptor is unable to interact with Gi/o but can signal through Gq/11. Additionally, my data do not support the suggestion of GnRH receptor-Gs interaction. Furthermore, I show that the GnRH Iinduced inhibition of cell growth is dependent on Gq/11, src and extracellular signal regulated kinase (ERK) but is independent of the activity of protein kinase C (PKC), Ca2+, jun-N-terminal kinase (JNK) or P38. Based on these findings and previous research within our group, I propose a mechanism whereby GnRH I may induce antiproliferation. Previous studies from our laboratory suggest that the GnRH receptor can adopt distinct active conformations in response to the binding of GnRH I and GnRH II. These data thus account for our hypothesis of ligand-induced selective signalling (LiSS). Given my previous results, I examined the ability of the GnRH receptor to couple to G12/13. My work indicates that the receptor can directly activate G12/13 and the downstream signalling cascades associated with this G protein family. Indeed, I provide evidence, in several cellular backgrounds, to suggest that GnRH receptor- G12/13-mediated signalling is involved in the regulation of GnRH-induced MAPK activity, SRE-driven gene transcription and cytoskeletal reorganisation. Furthermore, I propose a role for these G proteins in the transcriptional regulation of LHβ and FSHβ. Finally, I confirm previous results from our laboratory indicating that the GnRH receptor may interact with src Tyr kinase and show that GnRH I but not GnRH II may, independently of Gq/11, stimulate the Tyr phosphorylation and thus the activation of this protein. I propose that this differential signalling accounts for the distinct effects of GnRH I and GnRH II on cellular morphology and SREpromoted transcriptional activity. The research presented within this thesis provides evidence to refute published conclusions based on largely circumstantial experimental data, describes novel GnRH receptor signalling pathways and offers support for the concept of LiSS. It may assist in the development of new therapeutic compounds which selectively target one GnRH-mediated signalling pathway while bypassing others.

With approximately 8.5 million unwanted cats euthanized in the U.S. annually, convenient, cost effective methods of sterilization are greatly needed. Current spay/neuter techniques, such as surgery and hormonal intervention, are not satisfying this need due to their high cost, significant expertise required, and the need for feral cats to be collected and brought into clinics for treatment. The aim of this research is to develop a safe contraceptive vaccine that could be delivered to the feral cat population in bait without compromising non-feline species. Feline Herpes Virus (FHV) is a feline specific virus. The USDA has approved the immunization of cats with an attenuated, non-pathogenic strain of FHV expressing foreign antigens. In our research, we have partially replaced Glycoprotein I of FHV to express a fusion protein of Flagellin (FliC), Enhanced Green Fluorescent Protein (EGFP), and Gonadotropin Releasing Hormone (GnRH). FliC has been shown to stimulate a heightened antibody response when antigens are expressed as fusion proteins with it. GnRH, a major reproductive hormone responsible for the development of testes and ovaries in felines, is the target of our vaccine vector. Expression of EGFP will allow tracking of the viral vector. The expression of the fusion protein (FliC-EGFP-GnRH) is expected to stimulate an antibody and cell mediated immune response directed towards feline GnRH, which will provide an immunocontraceptive effect specific to cats.
Master of Science

To date, the pro-invasive role of the GnRH-GnRHR system has been demonstrated in several cell types including carcinoma cells. Placental expression of GnRH I, GnRH II, and their mutual receptor (GnRHR) is indicative of a potential mechanism(s) that occurs during the dynamic process of human placenta formation and differentiation, particularly during the development of an invasive phenotype of extravillous trophoblasts (EVTs). However, current studies haven’t completely revealed the role of GnRH in regulating invasive EVT function and the underlying mechanism(s) is not yet well-established. Dynamic reprogramming of cell adhesion and proteolytic machinery is frequently accompanied with cell invasive and angiogenic phenotypes. I hypothesized that GnRH could regulate trophoblast invasion and vascular remodeling via modulation of mesenchymal cadherins and matrix matelloproteinases (MMPs). In these studies, I have found that both GnRH forms could regulate N-cadherin and cadherin-11 expression distinctly by activating of transcription factors TWIST and c-FOS/c-JUN, respectively. Furthermore, I have demonstrated that GnRH I and GnRH II are capable of increasing MMP-2 and MMP-9 expression in EVT cells via up-regulation of the transcription factor RUNX2. Specific inhibition of TWIST/N-cadherin, c-FOS/c-JUN/cadherin-11 and RUNX2/MMP-2/MMP-9 in EVT cells attenuates both basal and GnRH-induced trophoblast invasion. Additionally, both forms of GnRH stimulate matrigel-mediated capillary-like network formation of trophoblastic cells and this phenomenon is also mediated by GnRH induced N-cadherin, cadherin-11, MMP-2 and MMP-9 expression. Collectively, our observations strengthen our hypothesis that GnRH is an important regulator of EVT cell behavior during implantation and placentation. These studies systemically described the underlying molecular mechanisms involved in GnRH induced adhesion molecule and proteolysis reprogramming.

Neuropeptides are well known to govern numerous biological functions and are found in all phyla studied to date. Probably the best known neuroendocrine system is the hypophyseal-portal system found in vertebrates, and one of the functions of this system is to mediate reproduction. Mammalian reproduction is controlled by a hormonal cascade which begins in distinct brain regions, namely the hypothalamus and the pituitary gland. Gonadotropin-releasing hormone (GnRH) is a neuropeptide typically produced in the hypothalamus. It is the key neuropeptide for initiating this cascade, and without it, reproduction cannot occur. Naked mole rats (Heterocephalus glaber) have a rigid social hierarchy. The "queen" is the most dominant female and is the only female who breeds. All aspects of reproduction are suppressed in other females in the colony: these "subordinates" are in a prepuberty-like state as they do not ovulate or display breeding behaviours. They are, however, not infertile, and are capable of rising to the breeding position. Since GnRH is the "master hormone" of reproduction, this study investigates its role in the socially-induced suppression of reproduction in female H. glaber. Brains of breeding (n = 7) and non-breeding (n = 5) female naked mole rats were compared to determine any differences in brain size, particularly in regions related to GnRH production. Noteworthy morphological and physiological transformations accompany the change from subordinate to dominant social status, including a significant increase in body length (Mann Whitney U test; p = 0.005, U = 0.000), body mass (Mann Whitney U test; p = 0.009, 1.000) and pituitary width and length (Mann Whitney U test; p = 0.028, U = 0.500 and p = 0.018, U = 0.000, respectively). Since little is known about the GnRH system in H. glaber, this study used immunocytochemistry to identify the distribution and abundance of GnRH neurons in the brains of both breeding and non-breeding females. GnRH neurons were found in the median eminence of the hypothalamus and in the anterior pituitary of both queens and subordinates, however in the brain of queen (n = 7) naked mole rats, there is a significantly larger area of immunoreactivity in comparison to the subordinate (n = 5) brain tissue (Mann Whitney U = 4.000, p = 0.030). This suggests that, in , subordinates, GnRH is inhibited at the level of production. The amino acid structure of the form of GnRH found in the brain of the naked mole rat is currently unknown, therefore a pilot study was carried out, using synthetic mammalian GnRH (mGnRH), mouse brain tissue and naked mole rat pituitaries and hypothalami, to examine and modify (where necessary) the methodologies used for neuropeptide extraction, purification and identification. A limited number of naked mole rats were available as source tissue (n = 4), therefore this study also tested whether it is possible to extract and purify an unknown neuropeptide from only a few mammalian samples. Training for reverse-phase liquid chromatography (RP-HPLC) was achieved by practicing the necessary methods with crude extracts prepared from stick insect (Carausius morosus) corpora cardiaca, which also served to compare vertebrate and invertebrate neuroendocrine systems. Synthetic mGnRH was used to demonstrate repeatability of the protocol and to set up suitable conditions for elution of mGnRH: mGnRH elutes at â 12 min when a solvent gradient of 32 % - 47 % B is applied. Synthetic mGnRH was also used to establish the amount of peptide required for accurate identification of GnRH by antigenicity tests (ELISA) and mass spectrometry. However, when extracts of mouse or naked mole rat brain matter were applied to this system, purification of GnRH was unconvincing as either there was insufficient material, or, some endogenous factor was masking the GnRH. Future studies would benefit from using molecular techniques as they require smaller amounts of source tissue. Alternatively, a larger amount of source tissue would be necessary in order to proceed with biochemical studies. While the impaired production of GnRH in subordinate naked mole rats seems to be linked to their prepuberty like state, it is unlikely that GnRH is the sole factor involved. Many other hormones (such as kisspeptin, gonadotropin-inhibiting hormone and neuropeptide Y) may influence GnRH and sexual maturity, and future studies would benefit from a multi-layered approach to investigate suppression of reproduction in naked mole rats.

GnRH is a decapeptide hormone, which plays a major role in the process of mammalian reproduction. It is synthesized by the hypothalamus and binds to its cognate receptor on the pituitary, to bring about the release of gonadotropins LH and FSH. The gonadotropin releasing hormone receptor belongs to the family of G-protein coupled receptors that are characterized by the presence of seven putative transmembrane regions linked by extracellular and intracellular loops. It is a glycoprotein made up of 327 amino acids. During the last several years cloning of this receptor from a number of species has provided considerable insight into the molecular basis of interaction between GnRH and its receptor. The GnRH receptor has been cloned and sequenced from a large number of mammalian species such as human, sheep, cow, rat, mouse, etc. GnRH receptor is known to be unique among the G protein coupled receptors by virtue of the fact that it lacks a C terminal tail which has been implicated in coupling to G-proteins in several seven transmembrane domain receptors. Other members of this G-protein coupled receptor family such as the Luetinising hormone receptor, Follicle stimulating hormone receptor contain the characteristic cytoplasmic tail of about 68-72 amino acids, which is believed to possess a plasma membrane targeting signal sequence. Mutation studies carried out revealed that this C terminal sequence may be important in membrane trafficking in other G protein coupled receptors, since mutant forms of the receptor were not expressed on the plasma membrane. In many G-protein coupled receptors, part of the cytoplasmic tail is important for desensitization and internalization. However, the GnRH receptor is an exception in that its G protein coupling and desensitization functions are dependent on regions of the GnRH receptor other than the carboxy terminal cytoplasmic domain. It has been well established that binding of GnRH to its cognate receptor induces conformational change and it is suggested that the entire extracellular loop and transmembrane region are involved in binding and signal transduction. It is pertinent to note in this connection, that the use of both polyclonal and monoclonal antibodies has contributed significantly to the understanding of the interactions between ligands and their cognate receptors. Recent studies have established that there are several extrahypothalamic sites of production of GnRH, which include testes, lymphocytes, human placenta, mammary gland etc. Of these the production of GnRH in the human placenta has attracted attention in view of the demonstration that the placental chorionic gonadotropin production (CG) is regulated by placental GnRH. Our laboratory has been investigating the role of GnRH in regulation of Chorionic Gonadotropin (CG) using both in vitro human placental villi system and pregnant bonnet monkey as models. One important and interesting observation that has been made in our studies as well as by several others is that the affinity of the placental GnRH receptor to its ligand is quite low compared to the pituitary receptor. Available evidence indicates that the hypothalamic and the placental GnRH are similar in structure and consequently the difference in the affinity could be attributed to the differences between the pituitary and the placental GnRH receptor. Considering this, it will be ideal and of interest to compare the GnRH receptor from the pituitary and placenta of a species in which both in vitro and in vivo studies can be carried out. For obvious ethical reasons, in vivo studies cannot be carried out with humans. Since very little information is available on the GnRH receptor in non-human primates, as a first step we undertook the task of characterizing the GnRH receptor from the bonnet monkey pituitary and production of antibodies to it, since all the studies carried out so far with antibodies to GnRH receptor have employed antibodies generated to a small stretch of peptide in the extracellular region. Thus the objective of the present study is to clone and express the GnRH receptor from the pituitary of the bonnet monkey {Macaco radiata), raise antibodies and to characterize them functionally. Chapter 1 provides a general review of information currently available regarding structure of GnRH and its receptor as well as the results of studies using antibodies directed to the GnRH receptor fragments. Chapter 2 deals with the partial cloning of the GnRH receptor from the pituitary of the bonnet monkeys by the technique of RT-PCR. We were able to amplify a PCR fragment of 959bp corresponding to the almost full-length GnRH receptor sequence. Southern blot analysis using the full length human pituitary GnRH receptor cDNA as the probe revealed that the 959 bp product was able to hybridize to the probe, confirming the authenticity of the PCR product. Restriction mapping with three different restriction enzymes also gave the expected pattern. Additional evidence was obtained by cloning of this PCR product into expression vector pGEX 5X-2 and sequencing a number of clones. The sequences obtained were then subjected to homology search with other known GnRH receptor sequences available in the Genebank. The sequence was found to be 97% homologous to the human pituitary GnRH receptor sequence and also showed a high degree of homology with the GnRH receptor from other species. Although antibodies have been raised to the GnRH receptor by immunizing rabbits with synthetic peptides corresponding to extracellular regions of the receptor, most of the antibodies have a very low affinity towards the native receptor. Also results of studies using these antibodies indicated that the peptide antibodies failed to recognize the native receptor. Initially we made efforts to express the full-length receptor in E.coli BL21 cells. However, since we were not successful in our attempts to express the full length, we resorted to express a smaller fragment which corresponded to amino acids 164-266, that encompassed one extracellular, two transmembrane and one intracellular domain. Before we proceeded ahead to express this fragment, the authenticity of this fragment was established by southern hybridization, restriction mapping as well as sequencing. This monkey pituitary GnRH receptor fragment corresponding to 315 bp was cloned in the expression vector pGEX 5X-2 and the protein corresponding to this region was overexpressed as a recombinant fusion protein in E.coli. BL21 plys S strain. Overexpression of the protein was induced using IPTG and the lysate was subjected to electrophoresis on a SDS-PAGE gel A signal corresponding to 37Kda, which is in agreement with the expected size (GST portion of the fusion protein plus the peptide) was observed following induction with IPTG. The overexpressed protein was found to be localized to the inclusion bodies, and this was purified from inclusion bodies by cutting out the band corresponding to the overexpressed protein from the preparative SDS-PAGE gels and the protein was eluted out by electroelution. Sera from the rabbits, which were immunized with the overexpressed protein, were checked for the presence of antibodies by ELISA, using the purified protein as the antigen. After ascertaining the presence of high titre antibodies in the sera of immunized animals, the serum was used to detect the presence of GnRH receptor in the membrane preparations from rat pituitary, monkey pituitary and human placenta using the technique of western blotting. A signal corresponding to 68Kda was found in all the cases and the specificity of this signal was established by preabsorption of the antisemrn with pituitary and placental membrane preparations, which resulted in decrease in the intensity of the signal. . The antiserum was also used to localize the GnRH receptor in different tissues such as first trimester and term human placenta, sheep pituitary, monkey placenta, human pituitary and rat prostate by the technique of immunotlourescence using the confocal microscope. The results of the above studies are presented in Chapter 3. Chapter 4 deals with the functional studies carried out using the antiserum to GnRH receptor in an in vivo system using male and female rats. As discussed earlier, all the reported studies on use of antibodies to GnRH receptor have employed a small region of the extracellular portion of the receptor for the production of antibodies. However, the antibodies in the present study have been directed towards a larger fragment, and considering this, it was of interest to evaluate the effect of these antibodies in in vivo as well as in vitro systems. Two approaches were used to evaluate the effect of antibodies, namely passive and active immunization i.e. administration of antiserum to GnRH receptor fragment raised in rabbits and also immunization with the overexpressed recombinant GnRH receptor protein. This study was carried out in both immature as well as adult male rats and also in the cycling female rats. Several parameters were monitored, which included various androgen dependent parameters in the male reproductive tissue i.e. body weight, testes weight as well as the weight of accessory sex organ-the prostate and also the fertility status. In the female rats the changes in the weight of the ovary, uterus, serum E2 and P4 were monitored. No effect on the body weight, testis weight or prostate weight was noticed in the treated animals compared to the controls. Furthermore, an indication that the hypothalamo-pituitary-gonadal axis was not compromised in the passively immunized animals was obtained from the observation that there was no decrease in the serum and testicular testosterone levels. In fact, there was a significant increase in the serum and testicular testosterone levels. This suggested the possibility that the antibodies are exerting a ßßstimulatory effect. To ascertain this possibility, two androgen dependent parameters namely the levels of mRNA for TGF ß, which is androgen repressed gene and Prostatein Cl, which is an androgen induced gene were monitored. It was observed that there was a significant increase in the steady state mRNA level of Prostatein Cl in GnRH antiserum treated animals and a corresponding decrease in TGFß mRNA levels. Active immunization study with injection of the recombinant protein was also carried out in adult male rats. All immunized animals responded to the immunization by producing high titre antibodies, the presence of which was detected by ELISA using the recombinant protein as the antigen. The results of the study revealed that there was no change in the body weight, testis weight or prostate weight. However, there was a significant increase in the serum and testicular testosterone levels compared to the control animals. Fertility studies indicated that all the animals were fertile. However, as in the case of passive immunization studies, an increase in the mRNA levels of Prostatein Cl was noted although the level of TGFß, which is an androgen repressed gene could not be monitored in this case due to the very high levels of endogenous androgens present in these animals. Thus it appears that the antibodies produced both in rabbits as well as in rats were stimulatory in nature probably indicating some specific characteristic of the region of the receptor to which the antibody has been raised. The results obtained in the present study are of significance considering the fact that studies using the antibodies to LH receptor and TSH receptor, both of which belong to the G-protein coupled family also report production of stimulatory antibodies. Active immunization studies using the GnRH receptor protein in the female rats also revealed that the antibodies were not compromising the hypothalamo-pituitary-gonadal axis. Accordingly, there was no decrease in the serum or ovarian levels of estradiol 17ß and progesterone and there was no difference in the ovarian weight. However, a significant decrease in the uterine weight and difference in the histology of the uterus of the immunized animals was observed. This is of significance, considering the fact that the presence of the GnRH receptor has been reported in the uterus also. In an attempt to develop an in vitro system to monitor the effect of GnRH receptor antibody, an in vitro incubation system with the human placental villi, which is known to produce both GnRH and hCG was standardized. Sensitive ELISA and RIA were developed for GnRH and hCG, respectively to monitor their levels.The results of the studies on the effect of addition of GnRH receptor antibody to the immunoreactive hCG levels in the placental incubation medium are presented in Chapter 5. In addition, advantage was taken of the report of the presence of the specific receptors for GnRH in the Leydig cells of the rats, to evaluate the effect of the GnRH receptor antibodies on the function of leydig cells. Results of studies in which the effect of addition of GnRH receptor antibodies on the testosterone production by purified rat Leydig cells were monitored revealed that there was no inhibitory effect. Finally in the Chapter 6, a general discussion and critical evaluation of the results obtained in the study, in light of similar studies reported in literature are presented.

Thesis (MSc)--Stellenbosch University, 2001.
ENGLISH ABSTRACT: The regulation of gonadotropin releasing hormone (GnRH) receptor numbers in the pituitary is a crucial control point in reproduction. Pituitary sensitivity to GnRH can be directly correlated with GnRH receptor levels, which can be regulated at transcriptional and post-transcriptional level. The proximal promoter of the mouse GnRH receptor gene contains two cis elements bearing the consensus sequence for a Steroidogenic Factor-l (SF -1) binding site. The distal site has previously been shown to be involved in basal and tissue-specific transcriptional regulation, whereas the function of the proximal site was not established. SF-I, a member of the nuclear receptor superfamily of transcription factors, is involved in the transcriptional regulation of a large number of genes involved in steroidogenesis and reproduction. The consensus SF-I binding site can serve as a binding site for several members of the nuclear receptor superfamily. The aim of this study was to investigate the binding of SF-I protein from the aT3-1 gonadotrope cell line to the two putative SF-I binding sites in the mouse GnRH receptor promoter in vitro, in order to provide supporting evidence for their functional roles in GnRH receptor gene regulation. It was shown by Western blotting that SF-I and Nur77, another nuclear receptor transcription factor, are both expressed in aT3-1 cells, in a manner that is influenced by cell culture conditions. Gel mobility shift assays using specific antibodies showed that both SF-I and Nur77 protein in aT3-1 nuclear extracts bind to both sites in a mutually exclusive fashion. As shown by competition assays using mutated versions of the two sites, Nur77 protein had different base pair requirements than that of SF-I protein for binding to the sites. Additionally, SF-I mRNA was shown by Northern blotting to be increased in aT3-1 cells in response to stimulation of the Protein Kinase A (PKA) pathway by forskolin. These results highlight unexpected degeneracy in so-called "consensus" nuclear receptor binding sites. Furthermore, since Nur77 protein is involved in the stress response of the hypothalamic-pituitary-adrenal (HPA) axis, the unexpected presence of Nur77 protein in a gonadotrope cell line has potentially important implications for cross-talk between the HPA and hypothalamic-pituitary-gonadal (HPG) axes.
AFRIKAANSE OPSOMMING: Daar bestaan 'n direkte verband tussen pituïtêre sensitiwiteit vir gonadotropien-vrystellingshormoon (GnRH) en GnRH-reseptorvlakke Die regulering van GnRH-reseptorvlakke op transkripsionele en post-transkripsionele vlak in die pituïtêre klier is belangrik by die beheer van voortplantingsfunksies. Die proksimale promotor van die GnRH-reseptorgeen in die muis bevat twee cis elemente met die konsensus volgorde vir 'n Steroidogenic Factor-l (SF-I) bindingsetel. Die distale element is betrokke by basale en weefsel-spesifieke transkripsionele regulering, maar die funksie van die proksimale element is nog nie vasgestel nie. SF-1 is 'n lid van die superfamilie van selkernreseptore en is betrokke by die transkripsionele regulering van gene verantwoordelik vir steroïedogenese en voortplanting. Die konsensus SF-I bindingsvolgorde kan dien as bindingsetel vir verskeie selkernreseptore. Ten einde 'n beter insig ten opsigte van die regulering van die GnRH reseptorgeen te verkry, is ondersoek ingestel na die binding van SF-I-proteïen, afkomstig van die aT3-1 pituïtêre gonadotroopsellyn, aan die twee moontlike SF-l bindingsetels in die GnRH-reseptor promotor, in vitro. Die Western-klad metode het getoon dat beide SF-l en Nur77, 'n ander selkernreseptor-transkripsiefaktor, in die aT3-1 sellyn uitgedruk word. Die uitdrukking is afhanklik van selkultuurtoestande. Elektroforetiese mobiliteitsessais met spesifieke antiliggame het getoon dat SF-l en Nur77 proteïene in aT3-1 selkernproteïenekstraksies eksklusief aan beide bindingsetels bind. Nur77 proteïen benodig ander basispare as SF-l proteïen om aan die bindingsetels te bind. Hierdie resultate dui op onverwagse degenerasie in sogenaamde "konsensus" selkernreseptor-bindingsvolgordes. Die Northern-kladmetode het ook getoon dat SF-l mRNA vlakke in aT3-1 selle styg wanneer die proteïenkinase A (PKA) pad gestimuleer word met forskolin. Aangesien Nur77 proteïen betrokke is by die stres-respons van die hipotalamus-pituïtêre klier-adrenale (HP A) aksis, hou die onverwagse teenwoordigheid van Nur77 proteïen in 'n gonadotroop-sellyn potensieel belangrike inplikasies in vir kommunikasie tussen die HPA-aksis en die hipotalamus-pituïtêre klier-gonadale (HPG) aksis.

The homogeneous assay format has been identified as having the potential to make an effective impact in the field of 'point-of-care or near patient testing. Homogeneous assays have the advantage that, by eliminating the need for multiple preparation steps, they can be rapid and easy to use in comparison with most solid-phase assay formats. However homogeneous assays tend to be generally less sensitive than their heterogeneous counterparts, giving results that are qualitative or at best semi-quantitative. This work presents a 'model' fluorescence-based homogeneous immunoassay for the detection of gonadotropin-releasing hormone, type-1, (GnRH-I) described by fluorescence spectroscopy and in particular time-resolved fluorescence techniques. In the model assay a new synthetic labelled 9-amino acid peptide, [des-pGlup1s]-LH-RH-Acp-FITC, is introduced to compete with GnRH-I for the two binding sites on the antibody 7B10.1D10. The core results demonstrate a photophysical characterisation of the binding of [des-pGlup1s]-LH-RH-Acp-FITC and 7B10.1D10 in homogeneous solution based on time-resolved fluorescence techniques. Specifically, values extracted from the plateau region of the time-resolved anisotropy decay curves are used to estimate the amount of free and bound [des-pGlup1s]-LH-RH-Acp-FITC and comment on the presence of interference processes in the assay. Furthermore, disruption to a system of [des-pGlup1s]-LH-RH-Acp-FITC bound to 7B10.1D10 by the addition of GnRH-I is described.

Bibliography: leaves 75-89.
The identification of multiple forms of gonadotropin-releasing hormone (GnRH) in a single species is becoming a common occurrence. The highly conserved chicken GnRH II is present along with one or two other GnRHs, composing a combination unique to particular species. This multifunctional peptide is widely distributed through the central nervous system and peripheral tissues. Also, endogenous GnRHs demonstrate distinct patterns of spatial expression within the brain, suggesting they may have separate functions. In addition to being the primary regulator of gonadotropin secretion in vertebrates, GnRH is also involved in the release of GH and prolactin and may fulfil a possible neuromodulatory role. GnRHs exert their actions through the stimulation of distinct GnRH receptors on pituitary gonadotrophs. The presence of multiple GnRH receptor subtypes has been demonstrated in several species and is likely to be a common characteristic of most vertebrates. This thesis describes the cloning and characterisation of GnRH receptors in two species of teleost fish, Haplochromis burtoni (cichlid) and Dania rerio (zebrafish). A type I GnRH receptor has previously been shown to exist in the cichlid. In the present study degenerate primers designed to extracellular loop three of the mammalian GnRH receptors were used to identify a second putative receptor subtype from cichlid (Haplochramis burtoni) genomic DNA. Furthermore, a near full-length cDNA, encompassing transmembrane domain 1 through to transmembrane domain 7 of the GnRH receptor, was cloned from cichlid RNA by reverse transcriptase PCR. This region of the receptor shares approximately 80% amino acid homology with corresponding regions of type III GnRH receptors previously identified in species of perciform fish. Partial sequences of a type IA and a type lB GnRH receptor have previously been identified in the zebrafish. Two sets of degenerate primers were used to elucidate the possible existence of a third receptor in the zebrafish using both genomic DNA and RNA. However, this strategy failed to result in the amplification of novel receptor subtypes in the zebrafish. Controversy surrounds the developmental origins of GnRH neurons and their temporal expression in relation to GnRH receptors. The zebrafish is a model organism, widely used for the study of reporter gene expression during development. Hence an attempt was made to isolate the zebrafish GnRH receptor genes using a genomic DNA library and identify the promoter regions for use as reporter genes in the study of GnRH and GnRH receptor expression during development. Southern blot analysis revealed six genomic clones with sequences homologous to zebrafish GnRH receptor cDNA. Comparison with genomic and cDNA sequences of other GnRH receptors revealed that those regions of the genomic clones that were sequenced only encoded exons 2 and 3. The presence of large introns in the GnRH receptor gene made it difficult to identify genomic clones containing the entire gene and the promoter region. The cloning of part of the zebrafish GnRH receptor genes will make their complete characterisation somewhat less problematic since an idea of their basic intron/exon structure has been obtained. Exons 2 and 3 of the zebrafish type IA and type IB GnRH receptor genes show a high degree of conservation when compared to the same regions of the goldfish type IA and type IB GnRH receptor cDNAs, demonstrating approximately 90% homology in both cases. In this study sequence information was obtained for the regions between transmembrane domains 4 and 7, and 3 and 7 of the zebrafish type IA and type IB GnRH receptor genes, respectively, and was subsequently used clone zebrafish GnRH receptor full-length cDNAs. This study describes the discovery of a type III GnRH receptor in the cichlid but suggests its presence may be restricted to only certain orders of teleost since a type III receptor was not identified in the zebrafish on this occasion. The information acquired from this study may help to reveal patterns, which relate the presence of particular GnRHs and GnRH receptors in single species to specific reproductive requirements.

Mutações inativadoras do receptor de GnRH (GNRHR) são a causa genética mais frequente de hipogonadismo hipogonadotrófico isolado (HHI) normósmico. Os genes envolvidos da patogênese do HHI, incluindo o GNRHR, estão associados a um amplo espectro fenotípico, variando de HHI parcial a completo. O atraso constitucional do crescimento e desenvovimento (ACCD) poderia constituir uma variante fenotípica leve do HHI. Neste estudo avaliamos a frequência de mutações no gene GNRHR em pacientes com HHI normósmico e ACCD, bem como correlacionamos o genótipo/fenótipo nesses pacientes. Além disso, avaliamos o efeito fundador de uma mutação do GNRHR (p.R139H) frequente na população brasileira com HHI normósmico. Para esse estudo, selecionamos 116 pacientes com HHI normósmico e 51 com ACCD. Um grupo de 130 indivíduos com desenvolvimento puberal normal foi utilizado como controle. A região codificadora do gene GNRHR foi amplificada por PCR e sequenciada. Análises in silico e in vitro foram realizadas nas duas novas variantes (p.V134G e p.Y283H). Três marcadores de microssatélites (D4S409, D4S2387, D4S3018) foram amplificados e analisados nos pacientes portadores da mutação p.R139H, familiares e controles. No grupo de HHI normósmico, nove mutações (p.N10K,p.Q11K, p.Q106R, p.R139H, p.C200Y, p.R262Q, p.Y284C, p.Y283H, p.V134G) foram identificadas em onze pacientes (9,5%). Entre as mutações identificadas no GNRHR, duas foram descritas pela primeira vez no estudo atual: p.Y283H e p.V134G, cuja análise in vitro demonstrou inativação completa do receptor. Em geral, uma boa correlação genótipo-fenótipo foi observada. Pacientes portadores de mutações inativadoras apresentavam HHI completo e mutações com perda parcial de função causavam HHI parcial, incluindo dois pacientes que evoluíram com reversão do hipogonadismo após reposição androgênica. Por outro lado, não houve diferença fenotípica entre os casos com e sem mutação do GNRHR. Análise de ancestralidade genética da mutação p.R139H demonstrou que todos os casos brasileiros apresentaram o mesmo haplótipo, sugerindo que a mutação p.R139H possui um ancestral comum na população brasileira. Por outro lado o caso familial proveniente da Polônia apresentou apenas um marcador em comum com as famílias brasileiras e estudos mais abrangentes seriam necessários para determinar a origem da mutação p.R139H em indivíduos não Brasileiros. Na casuística de ACCD apenas a mutação p.Q106R foi identificada no gene GNRHR em heterozigose em um paciente. Em conclusão, o GNRHR foi o gene mais comumente afetado, apresentando uma boa correlação genótipo-fenótipo, e deve ser o primeiro candidato para análise genética em HHI normósmico. Os resultados sugerem que a mutação p.R139H possui um ancestral comum na população brasileira. Mutações no GNRHR parecem não estar envolvidas na patogênese do ACCD
GnRH receptor (GNRHR) inactivating mutations are the most common genetic cause of normosmic IHH. The genes involved in the IHH, including GNRHR, have been associated with a large phenotypic spectrum, varying from partial to complete IHH. Constitutional delay of growth and puberty (CDGP) might represent a mild phenotypic variant of IHH. In this study we investigated novel variants and characterized the frequency and phenotype-genotype correlation of GNRHR mutations in normosmic IHH and CDGP patients. Additionally, we determined de cause of the recurrence of GNRHR p.R139H mutation in patients with normosmic IHH. We studied 116 patients with normosmic IHH and 51 with CDGP. The control group was composed by 130 adults with normal pubertal development. The coding region of GNRHR was amplified and automatically sequenced. The two novel variants identified (p.Y283H, p.V134G) were submitted to in silico and in vitro analysis. Three microsatellite markers (D4S409, D4S2387, D4S3018) were amplified by PCR and analyzed in the patients with the p.R139H mutation. In the CDGP group, the previously described mutation p.Q106R was identified in the heterozygous state in one boy. The p.Q106R mutation has been identified in heterozygous state in individuals with normal pubertal development and does not appear be involved on the CDGP phenotype in this patient. In the normosmic IHH group, nine variants were identified (p.N10K, p.Q11K, p.Q106R, p.R139H, p.C200Y, p.R262Q, p.Y284C, p.Y283H, p.V134G) in eleven patients (9.5%). In vitro analysis of the novel variants p.Y283H and the p.V134G demonstrated that both of them cause complete loss of function of the receptor. The founder effect study revealed that all the p.R139H affected Brazilian patients presented the same haplotype, suggesting that the this mutation has a common ancestor in the Brazilian population. Nevertheless the affected Polish family presented a different haplotype, with only one marker in common with the Brazilian families and further studies would be necessary to determine the origin of the p.R139H mutation in the European population. In conclusion this study demonstrated that GNRHR was the most commonly affected gene in normosmic IHH, with a good genotype-phenotype correlation, and should be the first candidate gene for genetic screening in this condition. The results of the founder effect study suggested that the p.R139H mutation has a common ancestor in the Brazilian population. Finally, mutations in the GNRHR do not appear to be involved in the pathogenesis of CDGP

Les neurones GnRH sont les principaux régulateurs de l'axe HPG de la puberté et de la fonction de reproduction normale. La puberté se caractérise par une maturation des circuits neuronaux. Cette période de maturation sexuelle dépend ainsi de profondes modifications de la régulation des connexions synaptiques et de la plasticité des neurones à GnRH. Les mécanismes responsables de ces modifications restent mal compris, mais ceux-ci pourraient dépendre de modifications structurales et de la maturation des neurones à GnRH. On a identifié une mutation dans le gène DMXL2, qui code pour la rabconnectin-3α (rbcn3-α), une protéine d'échafaudage liée à Rab3, qui joue un rôle dans la plasticité synaptique et le relargage de neurotransmetteurs. Rbcn3-α pourrait donc participer à la plasticité et la maturation des neurones à GnRH. Notre objectif a été de caractériser la rôle de rbcn3-a dans les neurones à GnRH. Rbcn3-α est exprimé dans des neurones GnRH. De souris knockout conditionnel de Dmxl2 dans les neurones entraine un retard pubertaire et une infertilité associés à une perte significative du nombre de neurones GnRH. Pour clarifier le rôle de rbcn3-α dans les perturbations neurodéveloppementales de GnRH, nous avons produit des souris Nes-Cre::Dmx12 et GnRH-Cre::Dmx12. On a ainsi montré que les neurones GnRH immatures ne peuvent pas être activés. De souris présentent un déficit de signalisation glutamatergique dans l'hypothalamus et un défaut du contrôles-circadiens de l'ovulation. Rbcn3- α participe à la maturation et l'activation des neurones à GnRH une étape indispensable à l'initiation de la puberté
Gonadotropin-releasing hormone (GnRH) neurons are the master regulatory output of the hypothalamus-pituitary-gonadal axis for pubertal onset. Puberty is characterized by the maturation of neuronal circuits where synaptic inputs change in the regulation of GnRH neurons to achieve pubertal onset. The mechanisms underlying these modifications remain elusive, but could be linked to the maturation of GnRH neurons. Deviations in the regulation of GnRH neurodevelopment cause pubertal delay and fertility defects. We discovered a complex neurodevelopmental disorder in human patients with a mutation in DMXL2, which encodes a vesicular protein rabconnectin-3α (rbcn3-α). Rbcn3-α is a scaffolding protein important for neuron plasticity and neurotransmitter release and can be a link between neuron plasticity and neurodevelopmental disorders with pubertal defects. Conditional neuronal Dmx12 knock-out mice (Nes::Cre;Dmx12loxp/wt) exhibitpubertal deficits, infertility, and a loss of GnRH neurons. We found loss of neuronal Dmx12 impedes GnRH neuron maturation, where immature GnRH neurons are unable to respond to potent stimuli compounded with disruptions in the circadian timed LH surge. These data indicate immature GnRH morphologies disrupts the functioning of GnRH neuronal networks. Moreover, Nes::Cre;Dmx12loxp/wt mice have decreased hypothalamic VGluT2 and increased NMDAR1 mRNA. Cre-dependent viral filling of GnRH neurons in GnRH::Cre ' Dmx12wt/wt mice showed rbcn3-a-containing terminals contact GnRH dendritic spines associated with VgluT2 terminals, and in the GnRH soma. We demonstrate rbcn3-a as a key regulator of pubertal onset and the maturation and activation of GnRH neurons

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
Infertility affects more couples and assisted reproduction techniques offer a possibility of treatment and the chance of having a child. Thus, the first attempt to ovulation induction is critical to the success of the cycle or even for future attempts is successful. Objective: To compare the protocols using GnRH agonist or antagonist for ovarian stimulation in normo-responders undergoing the first cycle of IVF/ICSI. Methods: we conducted a literature review on the history of ovulation induction controlled by medications. From the data available in the database of electronic medical records SISFERT used in the Laboratory of Human Reproduction (LabRep-HC-FM-UFG) a comparative retrospective observational study was conducted with 50 patients divided into two groups according to protocol: GnRH-agonist (leuprolide acetate 1 mg/day short protocol) or GnRHantagonist (Cetrorelix 0.25 mg/day), which received 150 IU/day of rFSH (follitropin alpha) and 250 µg of rhCG (alpha-coriogonadotrofina) in both groups. Results: Statistically significant differences were observed in the days of stimulation with rFSH, total dose of gonadotropin, days of use of GnRH, GnRH dose and total number of follicles (≥ 16 mm) on the day of the group rhCG GnRH agonist. There was no significant difference in other parameters, however, the number of oocytes retrieved was slightly higher in the GnRH agonist, but fertilization rate was higher in the GnRH-antagonist. Pregnancy rates and clinical chemistry were similar in both groups. Conclusions: although no significant differences in the results analyzed, the use of flexible antagonist protocol facilitates the handling and enables the patient using much lower doses of gonadotropins itself as the antagonist, reducing the cost of treatment when compared to the protocol with GnRH agonist.
A infertilidade afeta cada vez mais casais e as técnicas de reprodução assistida oferecem uma possibilidade de tratamento e a chance de ter um filho. Assim, a primeira tentativa de indução da ovulação é fundamental para o sucesso do ciclo ou, até mesmo, para que tentativas futuras sejam bem sucedidas. Objetivo: comparar os protocolos utilizando agonista ou antagonista do GnRH para estimulação ovariana em pacientes normo-respondedoras submetidas ao primeiro ciclo de FIV/ICSI. Métodos: foi realizada uma revisão da literatura sobre a história da indução da ovulação controlada por medicamentos. A partir dos dados disponíveis no banco de prontuários eletrônicos SISFERT utilizado pelo Laboratório de Reprodução Humana (LabRep–HC–FM–UFG), um estudo observacional retrospectivo comparativo foi conduzido com 50 pacientes distribuídas em dois grupos de acordo com o protocolo: GnRH-agonista (acetato de leuprolide 1 mg/dia protocolo curto) ou GnRH-antagonista (cetrorelix 0,25 mg/dia); e que receberam 150 UI/dia de rFSH (alfa-folitropina) e 250 µg de rhCG (alfa-coriogonadotrofina) em ambos os grupos. Resultados: foram observadas diferenças estatisticamente significativas nos dias de estimulação com rFSH, dose total de gonadotrofina, dias de uso do GnRH, dose total de GnRH e o número de folículos (≥ 16 mm) no dia do rhCG no grupo GnRH-agonista. Não houve diferença significativa nos outros parâmetros, no entanto, o número de oócitos recuperados foi ligeiramente maior no grupo GnRH-agonista, mas a taxa de fertilização foi maior no grupo GnRH-antagonista. As taxas de gravidez química e clínica foram similares nos dois grupos. Conclusões: embora não tenha havido diferenças significativas nos resultados analisados, o uso do protocolo flexível com antagonista facilita a manipulação pela paciente usuária e possibilita doses menores tanto de gonadotrofinas quanto do próprio antagonista, reduzindo o custo do tratamento quando comparado ao protocolo com agonista do GnRH

Objetivos: O objetivo deste estudo foi comparar os índices de Pulsatilidade (IP) e Resistência (IR) das artérias uterinas, o volume uterino e a espessura endometrial após o uso do Sistema Intra-uterino de Levonorgestrel (SIU-LNG) ou do agonista do GnRHa (GnRHa) em pacientes portadoras de endometriose pélvica. Pacientes e métodos: Setenta e nove mulheres voluntárias, com idade entre 18 e 40 anos, foram incluídas neste ensaio clínico comparativo, prospectivo, randomizado e controlado. Dezoito foram eliminadas do estudo baseadas nos critérios de exclusão, As 61 pacientes remanescentes foram divididas em dois grupos: 31 pacientes fizeram parte do grupo SIU-LNG (uma foi excluída antes da inserção por apresentar-se grávida) e 30 fizeram parte do grupo GnRHa. Foram submetidasa exame ultra-sonográfico transvaginal bidimensional no dia em que iniciaram o tratamento (inserção do SIU-LNG ou administração de uma ampola de 3,75 mg de GnRHa por via intra-muscular) e seis meses após, avaliando a espessura endometrial, o volume uterino e os IR e IP das artérias uterinas. Resultados: Ambos tratamentos promoveram redução da espessura endometrial (6.08±3.00mm para 2.70±0.98mm e 6.96±3.82mm para 3.23±2.32mm - média ±SD, grupo SIU-LNG e grupo GnRHa, respectivamente). O volume uterino teve redução no grupo usuário do GnRHa (86.67±28.38cm3 para 55.27±25.52cm3) sem alteração significativa nas usuárias do SIU-LNG (75.77±20.88cm3 para 75.97±26.62cm3). Em relação à ascularização uterina, notamos incremento dos IP das artérias uterinas em ambos os grupos (grupo SIU-LNG: artéria uterina direita de 2.38±0.72 para 2.76±0.99 (média ±SD) e artéria uterina esquerda 2.46±0.70 para 2.87±0.96, e grupo GnRHa: artéria uterina direita 2.04±0.59 para 3.12±0.98 eartéria uterina esquerda 2.24±0.59 para 3.15±0.89). Em relação ao IR das artérias uterinas, observamos incremento no grupo GnRHa em ambas artérias e somente na artéria uterina esquerda no grupo SIU-LNG (grupoSIU-LNG - artéria uterina direita de 0.85±0.08 para 0.88±0.07 e artéria uterina esquerda de 0.86±0.07 para 0.89±0.06, e grupo GnRHa: artéria uterina direita de 0.81±0.07 para 0.93±0.09 e artéria uterina esquerda 0.84±0.06 para 0.93±0.09). No entanto, ao compararmos as diferenças, a elevação foi significativamente maior nas usuárias do GnRHa. Conclusões: Ambos GnRHa e SIU-LNG promoveram redução na espessura endometrial e aumento no IP das artérias uterinas. Houve redução do volume uterino nas usuárias do grupo GnRHa, não se alterando no grupo SIU-LNG. Em relação ao IR, houve incremento em ambas as artérias nas usuárias de GnRHa e somente na artéria uterina esquerda nas usuárias do SIU-LNG.
Objectives:The objective of the present study was to compare the uterine arteries pulsatility index (PI) and resistence index (IR), uterine volume and endometrial thickness changes promoted by the use of the levonorgestrel intrauterine device (LNG-IUD) and the gonadotropin-releasing hormone analogue (GnRHa)in patients with endometriosis. Methods: Seventy nine women aged 18 to 40 years were included in this randomized controlled trial. Eighteen was excluded based on the exclusion criteria. The patients were randomly allocated in two groups: 31 women who used the LNG-IUD (since one became pregnant before insertion and wasexcluded) and 30 who used monthly GnRHa injections. They were submitted to a transvaginal two dimensional ultrasound scan on the day the treatment started and 6 months later, for the evaluation of uterine arteries PI, uterine arteries RI, uterine volume and endometrial thickness. Results: The use of LNG-IUD promoted an ndometrial thickness decrease (6.08±3.00mm to 2.7±0.98mm; mean±SD) as does the use of GnRHa (6.96±3.82mm to 3.23±2.32mm). The uterine volume decreased in the GnRHa group (86.67±28.38cm3to 55.27±25.52cm3), but not in the LNG-IUD group (75.77±20.88cm3 to 75.97±26.62cm3). Uterine arteries PI increased in both groups : Uterine arteries PI: LNG-IUD right uterine arterie 2.38 ± 0.72 to 2.76 ± 0.99 and left uterine arterie 2.46 ± 0.70 to 2.87 ± 0.96, and GnRHa right uterine arterie 2.04 ± 0.59 to 3.12 ± 0.98 and left uterine arterie 2.24±0.59 to 3.15 ± 0.89. Uterine arteries RI increased in both arteries in GnRHa and only in the left uterine arterie in the LNG-IUD :Uterine arteries RI : LNG-IUD right uterine arterie 0.85 ± 0..08 to 0.88 ± 0.07 and left uterine arterie 0.86 ± 0.07 to 0.89 ± 0.06, and GnRHa right uterine arterie 0.81 ± 0.07 to 0.93 ± 0.09 and left uterine arterie 0.84 ± 0.06 to 0.93 ± 0.09 . However, the increase was significant higher in the GnRHa group. Conclusions: Both GnRHa and LNG-IUD promoted an endometrial thickness decrease and an increase in the uterine arteries PI. The uterine volume decreased in women who used GnRHa, but not in those who used LNG-IUD.

La plante Phytolacca americana, plus communément appelée pokeweed, produit en grandes quantités des toxines protéiques appelées PAPs pour Pokeweed Antiviral Proteins. Ces toxines font partie de la famille des ribosomes inactivating proteins (RIPs). Elles bloquent la traduction en inactivant les ribosomes par dépurination de l'adénine 4324 dans une région très conservée de l'ARN ribosomal 28S. Ces RIPs confèrent une résistance virale aspécifique aux plantes qui les produisent suggérant leur participation aux mécanismes de défense antivirale. La grande sensibilité des ribosomes de la pokeweed vis-à-vis de la PAP implique l'existence de moyens de protection de la plante contre ses propres toxines. Nous avons mis en évidence au laboratoire, à l'aide d'anticorps monoclonaux, une forme complexée de PAP (PAPi) dans des extraits de feuilles de pokeweed. La PAPi possède un pI inférieur à celui de la PAP ce qui permet leur sépration par électrophorèse en condition non dénaturantes. La capacité de la PAP, sous sa forme complexée, à inhiber la traduction in vitro, est fortement réduite. Elle est cependant restaurée par dénaturation thermique du complexe. . .

Thesis (MSc)--University of Stellenbosch, 2007.
ENGLISH ABSTRACT: The gonadotropin-releasing hormone (GnRH) receptor is a G-protein-coupled receptor in the pituitary gonadotropes and is an important control point for reproduction. GnRH binds to the GnRH receptor (GnRHR) resulting in the synthesis and release of follicle stimulating hormone (FSH) and luteinizing hormone (LH). The sensitivity of the pituitary to GnRH can be directly correlated with GnRHR levels. The mouse GnRHR promoter contains three cis elements containing binding sites for steroidogenic factor-1 (SF-1), namely site 1 (-15/-7), site 2 (-244/- 236) and site 3 (-304/-296) as well as an activator protein-1 (AP-1)-like consensus sequence (TGAGTCA) at position –336/-330. While sites 1 and 2 and the AP-1 site have been previously shown to be involved in regulation of transcription of the mouse GnRHR (mGnRHR) promoter in some cell lines, the role of site 3 has not been previously investigated. This study investigated whether transcription of the mGnRHR gene is regulated by GnRH and glucocorticoids in the LβT2 gonadotrope pituitary cell line, and the role therein of site 3 and the AP-1 site and their cognate proteins, using a combination of in vitro protein- DNA binding studies and promoter-reporter assays. The role played by site 3 and the AP-1 site in basal transcription of the mGnRHR gene in LβT2 cells was the first area of investigation during this study. Luciferase reporter plasmids containing 600 bp of the mGnRHR promoter were used where the site 3 and AP-1 sites were either wild-type or mutated. Two constructs were prepared from the wild-type construct, i.e. wild type (LG), site 3 mutant (m3) and AP-1 mutant (mAP-1). Transfection of LG, m3 and mAP-1 plasmids into LβT2 cells was carried out to determine the effect of these mutations on the basal expression of the mGnRHR gene. Mutation of site 3 resulted in a 1.5 fold increase in the transcriptional activity of the mGnRHR promoter. This suggests that site 3 plays a role in the inhibition of basal transcriptional levels of the mGnRHR promoter in LβT2 cells. Mutation of the AP-1 site resulted in a 50% decrease in basal transcriptional levels of the mGnRHR promoter in LβT2 cells. This suggests that the AP-1 site is involved in positively mediating the basal transcriptional response of the GnRHR promoter in LβT2 cells. Experiments towards the understanding of the mechanism of the cis elements (site 3 and AP-1 site) on the mGnRHR promoter were carried out along with the role of protein kinase A (PKA) pathways, proteins involved and the effect of varying doses for varying times of GnRH, as well as the overexpression of PKA and the SF-1 protein. It was found that site 3 and the AP-1 site are not involved in the GnRH response. Results suggest that site 3 is partially involved in the PKA response in LβT2 cells. Site 3 can bind SF-1 protein as shown via competitive electrophoretic mobility shift assays (EMSA). When EMSA’s were performed on the AP-1 site the findings were that the c-Fos protein was not involved in the activation of the AP-1 site. A factor was found to bind to the AP-1 site, which did not require the intact AP-1 site, suggesting that it could be the c-Jun protein that binds to the AP-1 site under basal conditions. Another area that was investigated was whether the mGnRHR promoter can be regulated by dexamethasone (dex) either via the AP-1 site or site 3. A dose and time-dependent increase in promoter activity was observed with dex. This effect appears to require site 3 and the AP-1 site, as shown by the complete loss of response when these sites were individually mutated, consistent with a functional interaction between site 3 and the AP-1 site in LβT2 cells.
AFRIKAANSE OPSOMMING: Die gonadotropienvrystellings hormoon (GnRH) reseptor is ‘n G-proteïen-gekoppelde reseptor in die pituitêre gonadotrope en is ’n belangrike beheerpunt vir reproduksie. GnRH bind aan die GnRH reseptor (GnRHR) met die gevolg dat follikel stimulerende hormoon (FSH) en luteïeniserende (LH) gesintetiseer en vrygestel word. Die sensitiwiteit van die pituitêre klier vir GnRH kan direk met GnRHR vlakke gekorreleer word. Die muis GnRHR promotor bevat drie cis elemente met bindingssetels vir steroïedogeniese faktor 1 (SF1), naamlik setel 1 (-15/-7), setel 2 (-244/-236) en setel 3 (-304/-296) sowel as ’n aktiveerder proteïen 1 (AP-1) tipe konsensus sekwens (TGAGTCA) in posisie -336/-330. Terwyl setels 1 en 2 en die AP-1 setel voorheen getoon is om by die regulering van transkripsie van die muis GnRHR (mGnRHR) promotor in party sellyne betrokke te wees, is die rol van setel 3 nog nie vantevore bestudeer nie. In hierdie studie is ondersoek of die transkripsie van die mGnRHR geen deur GnRH en glukokortikoïede in die LβT2 gonadotroop pituitêre sellyn gereguleer word, en die rol van setel 3 en die AP-1 setel en hulle binders, deur gebruik te maak van in vitro proteïen-DNA bindings studies en promotor-verslaggewer essais. Die rol wat setel 3 en die AP-1 setel in basale transkripsie van die mGnRHR gene in LβT2 selle gespeel het, was die eerste onderwerp wat in hierdie studie bestudeer is. Lusiferase verslaggewer plasmiede wat die eerste 600 bp van die mGnRHR promotor bevat het en waarin setel 3 en die AP-1 setels óf wilde tipe óf gemuteer was, is gebruik. Two konstrukte is vanaf die wilde tipe konstruk berei, naamlik wilde tipe (LG), ’n setel 3 mutant (m3) en ’n AP-1 mutant (mAP-1). Transfeksie van LG, m3 en mAP-1 plasmiede in LβT2 selle is deurgevoer om te bepaal wat die effek van hierdie mutasies op die basale ekspressie van die mGnRHR gene was. Mutasie van setel 3 het ’n 1.5-voudige toename in die transkripsionele aktiwiteit van die mGnRHR promotor tot gevolg gehad. Dit suggereer dat setel 3 ’n rol in die inhibisie van die basale transkripsievlakke van die mGnRHR promotor in LβT2 selle speel. Mutasie van die AP-1 setel het tot ‘n 50% verlaging in basale transkripsievlakke van die mGnRHR promotor in LβT2 selle gelei. Dit suggereer dat die AP-1 setel betrokke is in die positiewe bemiddeling van die basale transkriptionele respons van die GnRHR promotor in LβT2 selle. Eksperimente wat gemik was om die meganisme van die cis-elemente (setel 3 en die AP-1 setel) op die mGnRHR promotor te verklaar, asook om die rol van proteïen kinase A (PKA) paaie, proteïene daarby betrokke en die effek van varieende dosisse vir verskillende tye van GnRH, sowel as die oorekspressie van PKA en die SF-1 proteïen, is deurgevoer. Dit is gevind dat setel 3 en die AP-1 setel nie betrokke by die GnRH respons is nie. Die resultate suggereer dat setel 3 gedeeltelik betrokke is by die PKA respons van LβT2 selle. Setel 3 kan SF-1 proteïen bind soos getoon deur kompeterence elektroforetiese mobiliteits verskuiwings essais (EMSA). As EMSA’s deurgevoer is op die AP-1 setel is bevind dat die c-Fos proteïen nie betrokke is in die aktivering van die AP-1 setel nie. ’n Faktor is gevind om aan die AP-1 setel te bind wat nie ’n intakte AP-1 setel vereis het nie, wat gesuggereer het dat dit die c-Jun proteïen kan wees wat aan die AP-1 setel onder basale omstandighede bind. ’n Ander area wat ondersoek is, is of die GnRHR promotor gereguleer kan word deur deksametasoon (dex) óf via die AP-1 setel óf via setel 3. ’n Dosis en tyds-afhanklike toename in promotor aktiwiteit is waargeneem met dex. ’n Vereiste vir hierdie effek blyk om die teenwoordigheid van setel 3 en die AP-1 setel te wees, soos aangetoon deur die totale verlies aan response as hierdie twee setels individueel gemuteer is, en wat weereens in ooreenstemming met die funksionele interaksie tussen setel 3 en die AP-1 setel in LβT2 selle is.

L’acquisition de la fertilité chez les mammifères est le résultat d’un long processus de développement et de maturation de l’axe gonadotrope. Cette fonction cruciale à la survie des espèces est orchestrée par une poignée de cellules localisées au niveau de l’aire préoptique hypothalamique chez le rongeur, sécrétant la gonadotropin-releasing hormon (GnRH). La GnRH stimule la sécrétion de LH et de FSH par l’adénohypophyse, qui stimulent à leur tour les gonades. Les cellules à GnRH naissent dans l’épithélium voméronasal pendant le développement embryonnaire et migrent le long des axones voméronasaux pour atteindre l’hypothalamus. A la naissance le système est entièrement en place, toutefois il subira une phase de maturation avant d’atteindre la puberté, signant le début de la fertilité. Chez l’homme, un défaut de sécrétion de GnRH peut conduire à un hypogonadisme hypogonadotrope idiopathique (IHH) caractérisé par une subfertilité et une puberté retardée voire absente, ou même à un syndrome de Kallmann. Dans une grande partie des cas ce défaut de sécrétion est lié à un défaut de développement prénatal et à une diminution du nombre de neurones à GnRH dans dans l’hypothalamus. Depuis peu, la grande famille des semaphorines, déjà connues pour leurs effets chimiotactiques dans certains types cellulaires, et en particulier la semaphorine3A (Sema3A) via son récepteur la Neuropilin-1 (Nrp1), a été décrite comme un facteur indispensable au développement du système à GnRH et décrit comme un « gène Kallmann ». Toutefois son rôle spécifique dans les cellules à GnRH reste à élucider. Le premier objectif de ma thèse a donc été de déterminer le rôle de l’expression du récepteur Nrp1 dans les neurones à GnRH. Le suivi de la maturation sexuelle des animaux Gnrh::cre, Nrp1loxp/loxp (qui n’expriment pas la Nrp1 exclusivement dans les neurones à GnRH) a révélé l’apparition d’une puberté précoce et d’un phénotype de surpoids en comparaison aux animaux contrôles, corrélé à une accumulation des cellules à GnRH dans l’aire préoptique. L’étude de l’embryogenèse du système à GnRH chez ces animaux a démontré une augmentation du nombre de cellules à GnRH pendant leur migration. Nos résultats obtenus in vivo et in vitro suggèrent que la signalisation Nrp1 a un impact sur la survie des neurones à GnRH, et qu’elle module la motilité des cellules en migration et influe leur positionnement dans le cerveau. Le deuxième objectif de ma thèse a été d’étudier le rôle de l’exocytose dépendante du calcium et donc de la neurosécrétion dans les neurones à GnRH sur leur développement. Le suivi de la physiologie d’animaux Gnrh::cre; iBot, dont l’exocytose dépendante du calcium est abolit par clivage de protéine VAMP2/synaptobrevin 2 dans le neurone à GnRH, a révélé l’apparition de deux phénotypes distincts selon la pénétrance du transgène : un groupe ayant une puberté normale et un poids comparable aux animaux contrôles, et un groupe ayant une puberté retardée voire inexistante associé à un surpoids. Ces derniers présentent un IHH, une augmentation du tissu adipeux périgonadique et une hyperleptinémie, alors que la distribution des neurones à GnRH dans le cerveau n’est pas altérée. Ces données mettent en évidence le fait que l’activité de neurosécrétion dans les neurones à GnRH ne serait pas nécessaire pour leur développement embryonnaire, mais qu’elle pourrait jouer un rôle dans le maintien de l’homéostasie énergétique.Ces deux études mettent en avant un lien étroit entre axe gonadotrope et métabolisme énergétique chez les mammifères et ont dévoilés de nouveaux mécanismes qui pourraient être impliqués dans la physiopathologie de la reproduction chez l’homme
Fertility in mammals is the result of a long development and maturation process of the hypothalamic-pituitary-gonadal axis. The reproductive function is orchestrated by a small population of neurons, located in preoptic area of hypothalamus in rodents, and releasing in a pulsatile manner Gonadotropin-releasing hormon (GnRH) in the portal blood vessels, where it is transported to the anterior pituitary gland. GnRH neuropeptide triggers synthesis and release of the gonadotropins LH and FSH, which in turn stimulates development and function of the gonads. GnRH neurons differenciate extracerebraly in the nasal placode and migrate from the vomeronasal organ to the forebrain along olfactory/vomeronasal nerves. At birth, the system is ready, however it will undergo a maturation phase before reaching puberty, signing the beginning of fertility. Deficiency in GnRH release can lead to idiopathic hypogonadotropic hypogonadism (IHH), characterized by a defect in sexual maturation and delayed or no puberty, or even to Kallmann syndrome when the IHH is associated with a deficit in the sens of smell. These phenotypes could be linked to a defect during GnRH neuron migration period and a decrease of GnRH cells located in hypothalamus after birth. Numerous studies have described the influence of different molecules on the migration of GnRH neurons. Recently, the semaphorin family, well known for its chemotactic effects in some cell types, and particularly the semaphorin3A (Sema3A), has been described by our laboratory as an essential factor for the guidance of GnRH neurons during embryogenesis, and characterized as a « Kallmann gene ». However, the role of Sema3A, and its specific receptor Neuropilin-1 (Nrp1) in GnRH neurons remains to be elucidated. The first objective of my thesis was to determine the role of the expression of Nrp1 in GnRH neurons. The analysis of sexual maturation in mice in which Nrp1 expression was selectively knocked out in GnRH neurons revealed a precocious onset of puberty and overweight compared to control littermates, correlated with an accumulation of GnRH neurons in preoptic area. The study of the development of the GnRH system during embryogenesis has shown an increased number of cells during migration. In vivo and in vitro data suggested the involvement of Nrp1 signaling pathway in the survival of GnRH neurons, the control of their motility during migration, and their final positioning in the brain.The second objective of my thesis was to study the role of Calcium-dependent exocytosis, and thus neurosecretion, in GnRH neurons on their development. The monitoring of Gnrh::cre; iBot animals, in which calcium-dependent exocytosis is abolished by cleavage of VAMP2/synaptobrevin2 protein in GnRH neurons, showed the distinction of two different phenotypes. A subpopulation of mice underwent normal puberty onset, with a similar bodyweight than control littermates, and the other one never reached puberty and developed overweight. The later animals exihibited IHH, increase of the volume of perigonadic fat tissue, and hyperleptinemia, with no alteration of GnRH neuron number and distribution. This data established that neurosecretion in GnRH neurons is not a prerequisite for their migration during embryonic development but revealed that it could play an important role in metabolic homeostasis.Together these two studies highlight an intriguing direct connection between GnRH neurons and energy metabolism in mammals as well as new mechanisms that could be implicated in reproductive physiopathology in human