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.

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.

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.

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.

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

Thesis (Ph. D.)--University of Virginia, 2008. / Includes bibliographical references. Also available in electronic form as viewed 2/16/2009.

Made available in DSpace on 2016-02-05T18:29:24Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-05-29. Added 1 bitstream(s) on 2016-02-05T18:33:27Z : No. of bitstreams: 1 000854973_20161205.pdf: 410109 bytes, checksum: 5ceaa1852fc805e9d99744c37a96f7bb (MD5) Bitstreams deleted on 2016-12-06T15:11:22Z: 000854973_20161205.pdf,. Added 1 bitstream(s) on 2016-12-06T15:12:04Z : No. of bitstreams: 1 000854973.pdf: 2773395 bytes, checksum: daf1de70009034e8b87b75a4cc4b7610 (MD5) / 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

Orientador: Maria Emilia Franco Oliveira / Coorientador: Pedro Paulo Maia Teixeira / Coorientador: Fábio Morato Monteiro / Banca: Pietro Sampaio Baruselli / Banca: Lindsay Unno Gimenes / Resumo: Devido a comum ocorrência de regressão prematura (RP) do corpo lúteo (CL) em novilhas após primeira ovulação (OV), os objetivos do estudo foram: 1) acompanhar a dinâmica lútea após indução da OV em novilhas Nelore pré-púberes e 2) determinar diferenças relacionáveis a funcionalidade dessa estrutura. Cinquenta e sete fêmeas (289,61±32,28 kg, ECC de 5,66±0,65 e 17,47±0,81 meses de idade) foram divididas em dois grupos de tratamento para indução da OV. No grupo GP4+GnRH foi utilizado dispositivo intravaginal de progesterona (P4) de 3º uso por 10 dias e, 48 horas após remoção, aplicado 0,02mg de acetato de buserelina (GnRH), e no grupo GGnRH foi utilizado somente o GnRH. Os CLs formados foram acompanhados pela ultrassonografia a cada dois dias até a sua regressão funcional (diminuição do sinal vascular do Doppler colorido e concentrações de P4 abaixo de 1 ng/mL), sendo determinado para cada dia o diâmetro, área, valores numéricos (VPN) e heterogeneidade dos pixels e percentual (%) de vascularização. A velocidade do pico sistólico, velocidade diastólica final, índice de resistência e o índice de pulsatilidade (IP) da artéria ovariana também foram determinados para cada avaliação, além da concentração sérica de P4. Essas características foram comparadas entre os tratamentos, funções dos CLs (duração normal ou regredido prematuramente), dias das avaliações e suas interações, utilizando o procedimento MIXED do programa SAS (p≤0,05). Três animais de cada tratamento não responderam ao ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Due to the common occurrence of premature regression (PR) of the corpus luteum (CL) in heifers after the first ovulation (OV), the aims of this study were to: 1) monitor the luteal dynamics after OV induction in prepubertal Nellore heifers, and 2) determine differences related to the functionality of this structure. Fifty-seven females (BW 289.61±32.28 kg, BCS 5.66±0.65 and 17.47±0.81 months old) were divided into two treatment groups for OV induction. In the group GP4+GnRH, an intravaginal progesterone (P4) device of 3rd use was used for 10 days and, 48 hours after its removal 0.02 mg of buserelin acetate (GnRH) was applied, and in the GGnRH group only GnRH was used. Formed CLs were monitored via ultrasonography every two days until functional regression (decrease of the vascular signal of color Doppler and serum P4 concentrations below 1 ng/mL), being determined for each day the diameter, area, numerical values (NV) and heterogeneity of the pixels, and vascularization percentage (%). The systolic and diastolic peak velocity, resistance and pulsatility index (PI) of the ovarian artery were also determined for each day in addition to the serum P4 concentration. These characteristics were compared between treatments, CLs functions (normal duration or prematurely regressed), days of evaluations and their interactions, using the MIXED procedure of SAS program (p≤0.05). Three animals from each treatment did not respond to the OV inductor (6/57=11%), which determined an ovulation ... (Complete abstract click electronic access below) / Mestre

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. / Science, Faculty of / Mathematics, Department of / Graduate