The neuropeptides arginine-vasotocin (AVT) and corticotropin-releasing hormone (CRH) are involved in the hypothalamic-pituitary-interrenal (HPI) axis. During stress, the HPI axis is activated and cortisol is released into the blood. In addition to their role in the HPI axis, AVT and CRH also have behavioural effects. The roles of AVT and CRH in stress responses and agonistic behaviour were studied in this thesis, using two different models. In the first model, two strains of rainbow trout (Onchorhynchus mykiss) divergent in stress-induced release of cortisol were investigated. This was done by observing behaviour and stress responses under different conditions. These strains were found to have divergent stress coping strategies based on the observed behaviour and levels of plasma cortisol. This divergence in behaviour could be associated with the CRH system, since the mRNA levels of CRH differed between the strains during stress. However, no differences between strains were observed in AVT or its receptor expressions. In the second model, non-selected rainbow trout were paired and the effect of intracerebroventricular (icv) injections of an active substance (AVT, CRH or the CRH related peptide Urotensin-I (UI)) on fights for dominance was investigated. One fish of the pair received the active substance icv and the other received saline icv. Fish receiving AVT became subordinate in accordance with the suggestion that AVT attenuates aggression in territorial vertebrates. Fish receiving CRH became subordinate whereas UI showed no effect on fights for dominance. Further, both CRH and UI induced an anxiety-related behaviour similar to non-ambulatory motor activity in rats. In addition, CRH appeared to affect the dopaminergic and serotonergic systems. In this thesis, it is suggested that CRH is involved in the behavioural modulation of the stress coping strategies in teleost fish. Further, AVT and CRH seem to act inhibitory on aggressive behaviour.

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 (Ph. D.)--West Virginia University, 2003. / Title from document title page. Document formatted into pages; contains vi, 187 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 156-183).

Reproductive senescence in females is a natural part of the aging process. However, the process by which it occurs, and the relative role of each level of the hypothalamic-pituitary-gonadal axis, remains largely unknown. The neural circuitry regulating the hypothalamic axis, including glutamate acting through N-Methyl-D-Aspartate receptors (NMDARs) on GnRH neurons, appears to be key to this process. The NMDAR is tetrameric and composed of an obligatory NR1 subunit together with NR2 subunits. The subunit composition determines the channel kinetics of the receptor and changes through the life span. This dissertation examines the physiological role of the NR2b subunit on LH pulsatile release and LH surge, both important for reproductive function. The expression of NR2b subunits in the anteroventral periventricular (AVPV) nucleus of the hypothalamus was also examined in aging rats. Experiment 1 showed that the NR2b-antagonist, ifenprodil, enhanced pulsatile LH release in estradioltreated females (both age groups). Experiment 2 showed that the LH surge in middle-aged animals was slightly accelerated and that results were dependent upon prior reproductive status of the animals. In Experiment 3, examination of the NR2b-immunoreactive cell population in young, middle-aged, and aged ovariectomized females given vehicle, estradiol, or estradiol with progesterone showed an age-associated decline in NR2b density. However, the immunofluorescent fraction volume of NR1 colocalized with NR2b increased with aging, and that of immunofluorescent fraction volume of NR2b increased with estradiol treatment. This is indicative of the amount of protein expressed in the AVPV. In total, NR2b cell density in the AVPV declines with age, but the amount of NR2b expressed in NR1-positive cells increases, suggesting a larger population of NR2b containing channels. This may translate to age-associated inhibition of GnRH/LH activity, which is relieved with blockade of NR2bcontaining NMDARs. Thus, this dissertation describes a novel way to examine the mechanism by which age-associated changes to neuromodulators of the HPG axis may affect the onset of reproductive senescence. / text

published_or_final_version / Zoology / Master / Master of Philosophy

The involvement of corticotropin-releasing hormone (CRH) and neuropeptide Y (NPY) in the pathophysiology of anxiety and anxiety-related disorders is well established. The objective of this study is to explore the genetic variations in the CRH and NPY genes in a well-documented behavioral animal model, the vervet monkey (Chlorocebus aethiops sabaeus), in order to uncover a possible association between these polymorphisms and behavioral traits quantitatively extracted following analysis of social behavior and responses to novelty challenges. / The vervet CRH and NPY genes were amplified and sequenced; the priority was given to the regions expanding from -1kb upstream of the transcription initiation site (where most of the regulatory elements are found in both genes) through the second exon. / Polymorphism discovery analysis revealed the presence of 9 vervet CRH SNPs and 9 vervet NPY SNPs; the SNPs are relatively evenly distributed across the regions covered. An association between one intronic NPY SNP and "defensive aggression" was detected. / These results are coherent with other reports implicating NPY in defensive aggressive behavior, and support the notion that fear responses are fundamental behavioral traits for the dissection of anxiety.

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.

In temperate zones most breeds of goats are anestrous and anovulatory during spring and summer, but start cycling as day length decreases during the fall. In tropical zones, indigenous goats, such as the Pygmy and the Nigerian Dwarf, tend to cycle throughout the year. Some studies have indicated that dwarf breeds become more seasonal when they are raised in temperate zones, while others maintain that they are capable of breeding throughout the year. In this study, Pygmy and Nigerian Dwarf goats became more seasonal in Quebec. The majority of the animals were cycling during December and February, but a significant proportion exhibited anestrus during October, May, and June. Several hormones, including prostaglandins (PG), progestagens, and gonadotropins (eCG, FSH, GnRH, hCG), have been used for the control and synchronization of estrus and ovulation in goats, but limited research has been conducted with dwarf breeds. In this study, dwarf goats were synchronized in November, March, and July with a 10-day MAP sponge coupled with 125 mug cloprostenol i.m. 48 h before sponge removal and 300 IU eCG i.m. at sponge removal. A seasonal shift was detected in the intervals to the onset of estrus, the LH surge, and ovulation following sponge removal. These intervals were shorter in November and July than in March (P < 0.05). The intervals between the onset of estrus and the LH surge and between the LH surge and ovulation were found constant throughout the different seasons. The administration of 50 mug GnRH at 24 h after sponge removal improved the timing and synchrony of the LH surge and ovulation in dwarf goats (P < 0.05). The knowledge acquired from this research may serve to improve reproductive efficiency in dwarf goats by facilitating the determination of an optimal time for breeding, artificial insemination, and oocyte and embryo recovery.

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

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.