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Sex steroids, gonadotropins, and effects on the immune response in maturing spring Chinook salmon (Oncorhynchus tshawytscha)Slater, Caleb H. 31 October 1991 (has links)
Graduation date: 1992
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Alteration of N-linked oligosaccharide structures of human chorionic gonadotropin β -subunit by disruption of disulfide bondsMIZUOCHI, TSUGUO, NAKATA, MUNEHIRO, BOIME, IRVING, TOMODA, YUTAKA, KIKKAWA, FUMITAKA, FURUHASHI, MADOKA, SUGANUMA, NOBUHIKO, MORIWAKI, TAKAYUKI 02 1900 (has links)
名古屋大学博士学位論文 学位の種類 : 博士(医学)(論文) 学位授与年月日:平成9年2月28日 森脇崇之氏の博士論文として提出された
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Studies On Cloning And Characterization Of GnRH Receptor From The Pituitary Of Bonnet Monkey (Macaca Radiata) And Functional Studies With The Antiserum To GnRH ReceptorSantra, Sumana 01 1900 (has links)
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.
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Modeling electrical spiking, bursting and calcium dynamics in gonadotropin releasing hormone (GnRH) secreting neuronsFletcher, Patrick Allen 11 1900 (has links)
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.
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Untersuchungen zur Nutzung von Altrenogest (Regumate®) und Gonadotropinen zur Zyklussteuerung von Alt- und Jungsauen mit negativem TrächtigkeitsbefundBeckjunker, Jochen 30 May 2007 (has links) (PDF)
In der vorliegenden Arbeit sollte die Wirksamkeit von Altrenogest (Regumate®) und Gonadotropinen bzw. des GnRH-Analogons D-Phe6-GnRH im Rahmen eines Verfahrens zur Brunst- und Ovulationssynchronisation bei besamten, als ingravid detektierten Jung- und Altsauen überprüft werden. Die Untersuchungen wurden an insgesamt 265 Jung- und 542 Altsauen vorgenommen. Die Sauen wurden im Rahmen der ultrasonografischen Trächtigkeitskontrolle, die zwischen den Tagen 21 und 35 nach der ersten künstlichen Besamung durchgeführt wurde, als ingravid detektiert und einer von drei verschiedenen Versuchsgruppen (VG) zugeteilt: VG 1 (n = 490): Applikation von 4 ml Regumate®/Tier/Tag oral über 15 Tage; einmalige subkutane (s.c.) Injektion von 1.000 IE PMSG/eCG 24 Stunden nach letztmaliger Regumate®-Gabe; i.m. Applikation von 500 IE hCG 78 bis 80 h nach PMSG/eCG zur Induktion der Ovulation; VG 2 (n = 135): identisch zu VG 1, aber Gabe von 5 ml Regumate® und 800 IE PMSG; VG 3 (n = 182): identisch zu VG 2, aber Injektion von 50 µg D-Phe6-GnRH zur Induktion der Ovulation. Die Sauen wurden jeweils zweimal im Abstand von 24 und 40 Stunden nach hCG bzw. D-Phe6-GnRH künstlich besamt. Der Erfolg der Behandlungen wurde anhand der sonografischen Untersuchung der Ovarien kontrolliert. Untersuchungen erfolgten zum Zeitpunkt der Trächtigkeitsuntersuchung, am Ende der Regumate®-Behandlung, unmittelbar vor der ersten und unmittelbar nach der zweiten künstlichen Besamung. Trächtigkeits- (TR) und Abferkelrate (AFR) sowie Anzahl insgesamt und lebend geborener Ferkel wurden dokumentiert. Als jeweilige Kontrolltiere dienten Jung- bzw. Altsauen, die brunst- und ovulationssynchronisiert wurden, zur Erstbesamung anstanden und zeitgleich wie die Tiere der Versuchsgruppe besamt wurden. Die Auffindungsrate der Ovarien betrug 88,9 % zum Zeitpunkt der Trächtigkeitsuntersu-chung (TU) und 98,3 % am Ende der Regumate®-Behandlung. Während der Besamungen konnten bei allen untersuchten Tieren die Ovarien dargestellt werden. Zum Zeitpunkt der TU wiesen die Sauen überwiegend Corpora lutea (CL; 56,3 %; p < 0,05) auf. Der zweithäufigste Befund waren Follikel von 2-6 mm Durchmesser (F2-6; 27,7 %; p < 0,05). Durch Altrenogest konnte das Follikelwachstum effektiv gehemmt werden, ohne die spontane Luteolyse zu beeinträchtigen. Mehr als 84 % der Tiere wiesen am Ende der 15-tägigen Behandlung mit Regumate® F2-6 auf. Altsauen, die 4 ml Regumate® erhielten, hatten häu-figer als die mit 5 ml behandelten Tiere polyzystisch degenerierte Ovarien (POD). Zahlrei-che Altsauen, vor allem solche mit periovulatorischen Funktionsgebilden zum Zeitpunkt der Trächtigkeitsuntersuchung (p < 0,05), wiesen zudem CL am Ende der Behandlung auf (16,3 %). Jungsauen ovulierten unabhängig von der ovulationsauslösenden Substanz überwiegend zwischen beiden Besamungen (p < 0,05), während Altsauen zu gleichen Anteilen zwischen den Besamungen und nach der zweiten Besamung ovulierten. Die ovulationsauslösende Substanz, d.h. hCG bzw. GnRH, hatte weder bei Jung- noch bei Altsauen einen Einfluss auf das Ovulationsverhalten. Der Ovulationszeitpunkt war ohne Einfluss auf die Trächtig-keitsrate. Altsauen, die D-Phe6-GnRH erhielten, wurden häufiger tragend als Tiere, die hCG erhielten (p < 0,05). Reziprokes Ergebnis erzielten die Jungsauen (p < 0,05). Kon-trolltiere wiesen Trächtigkeits- bzw. Abferkelraten auf, die bis zu 15,6 % (TR; p < 0,05) bzw. 16,8 % (AFR) höher als die der Versuchstiere waren, da vor allem die Altsauen, nicht aber Jungsauen aller drei Versuchsgruppen schlechtere Fruchtbarkeitsleistungen erzielten. Aus den Ergebnissen ist zu schlussfolgern, dass die kombinierte Anwendung von Altrenogest (Regumate®) und Gonadotropinen bzw. GnRH-Analogon D-Phe6-GnRH zur Brunst- und Ovulationssynchronisation bei besamten, als ingravid detektierten Jung- und Altsauen geeignet ist. Ausgehend von den Ergebnissen ist zu empfehlen, Sauen mit 5 ml Reguma-te®/Tier/Tag zu behandeln (Zyklusblockade). Ein Applikationsintervall von 15 Tagen ist ausreichend. Werden 800 IE PMSG/eCG 24 Stunden nach letztmaliger Regumate®-Applikation verabreicht, kann mit zuverlässiger Stimulation des Follikelwachstums gerechnet werden. Jungsauen sollten 500 IE hCG 78 bis 80 Stunden nach der PMSG/eCG-Gabe erhalten. Bei Altsauen sind dazu 50 µg des GnRH-Analogons D-Phe6-GnRH emp-fehlenswert. Die transkutane Ultrasonografie ist ein geeignetes Verfahren zur Darstellung von Ovarien und ovarieller Funktionskörper.
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Modeling electrical spiking, bursting and calcium dynamics in gonadotropin releasing hormone (GnRH) secreting neuronsFletcher, Patrick Allen 11 1900 (has links)
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.
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Mammalian cell growth and proliferation mediated by the gonadotropin-releasing hormone (GnRH) receptor : role of novel interacting protein partnersMiles, Lauren E. C. January 2005 (has links)
[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.
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Diurnal and estradiol-dependent regulation of the neuroendocrine signal for ovulation /Christian, Catherine Anne. January 2008 (has links)
Thesis (Ph. D.)--University of Virginia, 2008. / Includes bibliographical references. Also available in electronic form as viewed 2/16/2009.
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Investigating the mechanism of transcriptional regulation of the gonadotropin-releasing hormone receptor (GnRHR) gene by dexamethasone /Von Boetticher, S. January 2008 (has links)
Thesis (MSc)--University of Stellenbosch, 2008. / Bibliography. Also available via the Internet.
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Ovarian cysts in dairy cattle : importance of serum LH concentrations in maintenance of cysts and expression of mRNAs for steroidogenic enzymes and gonadotropin receptors /Calder, Michele D. January 1998 (has links)
Thesis (Ph. D.)--University of Missouri-Columbia, 1998. / Typescript. Vita. Includes bibliographical references (leaves 110-136). Also available on the Internet.
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