The purpose of this experiment is to determine if immature female white mice will react to the gonadotropic hormone from the anterior pituitary of several species of fish. If they can be shown to do so, a second purpose is to establish an assay unit for this fish gonadotropin in order that it may be used with predictable results in inducing extra-seasonal spawning for fresh-water conservation practices.

Fertility is dependent on the precisely orchestrated communication of an array of effectors within the reproductive axis, all of which impinge on gonadotropin-releasing hormone (GnRH) neurons. A novel reproductive inhibitor was identified in avian species and growing evidence suggests that the functional mammalian homologue, RFamide-related peptide-3 (RFRP-3 or GnIH) can inhibit GnRH neuronal activity and gonadotropin release. To date, the regulation and effects of RFRP-3 at the hypothalamic level are poorly understood. We established an Rfrp-expressing neuronal cell model to investigate the mechanisms of transcriptional regulation of the genes for RFRP and the RFRP receptor, GPR147 by dexamethasone and estradiol. We show that the RFRP system is a direct target for stress-associated transcriptional regulation. Further, employing a novel GnRH-secreting cell line, we report that GnRH neurons express Gpr147 and RFRP-3 represses the transcription of GnRH. These data further our understanding of the level and regulatory effects at which RFRP-3 modulates reproduction.

Fertility is dependent on the precisely orchestrated communication of an array of effectors within the reproductive axis, all of which impinge on gonadotropin-releasing hormone (GnRH) neurons. A novel reproductive inhibitor was identified in avian species and growing evidence suggests that the functional mammalian homologue, RFamide-related peptide-3 (RFRP-3 or GnIH) can inhibit GnRH neuronal activity and gonadotropin release. To date, the regulation and effects of RFRP-3 at the hypothalamic level are poorly understood. We established an Rfrp-expressing neuronal cell model to investigate the mechanisms of transcriptional regulation of the genes for RFRP and the RFRP receptor, GPR147 by dexamethasone and estradiol. We show that the RFRP system is a direct target for stress-associated transcriptional regulation. Further, employing a novel GnRH-secreting cell line, we report that GnRH neurons express Gpr147 and RFRP-3 represses the transcription of GnRH. These data further our understanding of the level and regulatory effects at which RFRP-3 modulates reproduction.

[Truncated abstract] Alzheimer’s disease (AD) is a progressive neurodegenerative disease which manifests clinically as personality changes and global cognitive decline resulting in a loss of function, ultimately leading to death. Whilst causal genetic mutations have been identified, accounting for a small proportion of familial cases, the vast majority of all AD cases are late onset and idiopathic. However, a number of risk factors have been identified, including age associated changes in the reproductive hormones – estrogen and the gonadotropins. Previous in vitro and in vivo studies have implicated both estrogen and the gonadotropins in the regulation of the neurotoxic beta amyloid (Aß) peptide, accumulation of which is thought to be a key pathogenic event in the development of AD, but the role of these hormones in the etiology and pathogenesis of AD remains contentious. The aim of this thesis was to further understanding of the role of female reproductive hormones in modulating susceptibility to AD. The role of menopausal hormone dysregulation in behavior, cognitive decline and Aß-related neuropathology was examined in vivo in 4 studies using animal models of AD and menopause. The first two studies used a mouse model of AD expressing a human PS1 mutation (PS1KI) to examine the effects of ovariectomy as a model of menopause on cognition and neuropathology. Ovariectomy was found to selectively impair learning on a spatial working memory task without affecting working memory recall or reference memory performance. However, this cognitive impairment was not associated with any changes in Aß accumulation or oxidative stress. ... However, these findings cannot explain the lack of effect of estrogen supplementation on Aß levels. It is possible that supra-physiological doses of estrogen are necessary to yield anti-amyloidogenic and anti-oxidative benefits in ovariectomized sheep. It is becoming clear that the relationship between hormone changes at menopause and risk of AD may be more complicated than previously conceived. This study has begun to tease apart the relative contributions of estrogen and the gonadotropin hormones in the modulation of Aß, accumulation of which may confer susceptibility to AD. The findings presented indicate that the gonadotropins may play an important role in the regulation of AD-related behavior and cognition. The observed functional effects of the gonadotropins may also have implications for our understanding of behavioral and cognitive changes occurring during reproductive events. Based on the evidence presented here, combined with previous literature, it is clear that both estrogen and the gonadotropins are involved in the modulation of Aß accumulation, however, elucidation of the circumstances necessary to elicit these effects and their clinical relevance to humans will require further investigation. These findings contribute to a more sophisticated understanding of the post-menopausal hormonal milieu, recognizing the role of the gonadotropin hormones and that gonadal estrogen depletion does not necessarily result in brain estrogen depletion.

No description available.

No description available.

Corpus luteum (CL), a transient endocrine structure formed from the ruptured ovarian follicle after ovulation, secretes progesterone (P4) that is essential for establishment and maintenance of pregnancy in mammals. The biosynthesis and secretion of P4 from CL depends, in general, on trophic hormones of the anterior pituitary gland and on hormones or factors originating from ovary, uterus, embryo and placenta. The structure and function of CL tissue is regulated by intricate interplay between two types of factors, namely, the luteotrophic factors, which stimulate CL growth and function, i.e., P4 secretion, and the luteolytic factors, which inhibit CL function and lead to luteal regression. In monoovulatory species such as higher primates and bovines, a striking diversity in the regulation of CL function exists not only between species, but also within the species during different stages of the luteal phase. In higher primates, unlike other species, one of the important characteristics of CL regulation is that, during non-fertile cycle, circulating LH appears to be the sole trophic factor responsible for maintenance of its function, and during fertile cycle, chorionic gonadotropin (CG), an LH analogue, originating from placenta maintains CL function. In higher primates, the role/involvement of luteolytic factors during luteolysis remains elusive. On the other hand, in the bovine species, the role/involvement of luteolytic factor, prostaglandin (PG) F2α during luteolysis is well established. It should be pointed out that in both the species, the mechanism of luteolysis is still poorly understood and the work presented in this thesis attempts to address these lacunae. Further, in bovines, studies have been carried out to examine potential trophic factor(s) responsible for the maintenance of CL function. Chapter I provides an extensive review of literature on CL structure and function with emphasis on factors that influence its growth, development, function and demise in primates and bovines. In Chapter II, employing bonnet monkey (Macaca radiata) as the representative animal model for higher primates, various studies have been conducted to examine the role of molecular modulators involved in regulation of CL function, particularly during spontaneous luteolysis. Although, it is well established that LH is essential for the maintenance of CL function in higher primates, the mechanism(s) responsible for the decline in serum P4 levels at the end of non-fertile cycles, without a concomitant change in circulating LH milieu, remains to be addressed. Several experiments have been conducted to examine the component(s) of luteotrophic (LH/CG) signaling that is/are modulated during luteolysis in the bonnet monkey CL. To understand the relative lack of responsiveness of CL to the circulating LH during the late luteal phase, LH/CG receptor (R) dynamics (expression of LH/CGR and its various transcript variants) was examined throughout the luteal phase and during different functional states of the monkey CL. The results indicated presence of LH/CGR mRNA, its transcript variants and functional LH/CGR protein in the monkey CL on day 1 of menses. Moreover, the functionality of receptors was tested by confirming the biological response of the CL to bolus administration of exogenous LH preparations, which eventually suggested factor(s) downstream of LH/CGR activation to account for the decline in CL function observed during non-fertile cycle. Studies have been conducted to identify molecular modulators that would selectively exploit intraluteal processes to regulate trophic signaling pathways that are critical to the control of luteal function. Immunoblot and qPCR analyses were carried out to examine presence and activation of Src family of kinases (SFKs) and cAMP-phosphodiesterases (PDEs) during various functional states of CL. The results revealed an increased activation of Src (phosphorylated at Tyr 416) during spontaneous and PGF2α/CET-induced luteolysis that may participate in the regulation of cAMP levels in part by increasing the cAMP-PDE activity observed during spontaneous luteolysis. This observation raised the question on the possible mechanism by which CG, an analog of pituitary LH, rescues CL function during early pregnancy. Thus, subsequent experiments involving LH/hCG administration in CET-treated animals as well as simulated early pregnancy animal model were conducted and the results revealed that, a bolus of LH/hCG decreased Src activation and cAMP-PDE activity accompanying a momentous increase in cAMP levels in both these models that further led to a concomitant increase in P4 secretion. Although the mechanisms of action of LH/CG involve modulation of a number of signaling pathways in the CL, by far, the results from various experiments suggested that it leads to activation of Src kinase and cAMP-PDE, thus causing inhibition of various elements of the primary signaling cascade- AC/cAMP/PKA/CREB during spontaneous luteolysis. One of the consequences of activation of Src kinase and cAMP-PDE was the regulation of expression of genes associated with steroidogenesis and it was observed that expression of SR-B1, a membrane receptor associated with trafficking of HDL-CE into the luteal cells, was lower in the regressed CL. The results taken together suggest that the decrease in responsiveness of CL to LH milieu during non-fertile cycles is not associated with changes in LH/CGR dynamics, but, is instead coupled to the activation of Src kinase and cAMP-PDE, inhibition of molecules downstream of LH signaling, and a decrease in the SR-B1 expression that regulates cholesterol economy of the luteal cell, and in turn, P4 secretion. The control of primate CL function appears to be dominated by the luteotrophic factors (LH/CG) over the luteolytic factors, since the process of luteal regression was overcome by administration of LH/CG. Further, in the primate CL, the molecular modulators of LH/CG signaling (Src kinase and PDE) are maintained in the repressed state by the luteotrophic factor LH/CG for maximum steroidogenic function. In contrast, in non-primate species, without invoking a role for the luteotrophic factor, essentially the synthesis and secretion of luteolytic factor, PGF2α, from the uterus is kept in check during pregnancy by the trophoblast derived IFN- and thus allowing CL to continue to function that is essential for maintenance of pregnancy. In the bovine species, the mechanism of PGF2α-induced luteolysis that involves a change in expression of genes associated with various processes of cellular function is poorly understood. Experiments were conducted utilizing buffalo cows (Bubalus bubalis) as a model system, to determine temporal changes in the global gene expression profile of the CL in response to PGF2α treatment. For this purpose, CL tissues were collected on day 11 of estrous cycle without treatment (designated as 0 h) and at 3, 6 and 18 h post PGF2α treatment for various analyses. Global changes in gene expression pattern in the CL were investigated employing Affymetrix GeneChip bovine genome array and the results are presented in Chapter III. The hybridization intensity values obtained by microarray analysis were subjected to R/Bioconductor tool. Following the application of highly stringent statistical filters to eliminate false positives, a set of differentially expressed genes were identified. The differentially expressed genes were further classified based on a fold change cut-off filter of ≥2, and the analysis revealed 127 genes to be differentially expressed within 3 h of PGF2α administration, of these 64 and 63 genes were up-regulated and down-regulated, respectively. Analysis of microarray data at 6 h post PGF2α administration revealed 774 genes to be differentially expressed, of which 544 genes were up-regulated, while 230 genes were down-regulated. The microarray analysis performed on CL tissues collected at 18 h post PGF2α administration showed that out of the total 939 differentially expressed genes, 571 genes were up-regulated, while 368 genes were down-regulated. Analysis of the ontology report for the biological processes category showed that initially in response to PGF2α administration, genes regulating steroidogenesis, cell survival and transcription were differentially regulated in the CL, but at later time points, differential expression of genes involved in apoptosis, PGF2α metabolism, tissue remodeling and angiogenesis was observed. Further, involvement of molecules downstream of LH/IGF-1 activation was investigated and the results obtained indicated that PGF2α interfered with the LH/IGF-1 signaling since the expression of LH/CGR, GHR and pAkt were down-regulated following PGF2αadministration. Furthermore, the functional luteolysis observed post PGF2αadministration appeared to be due to an interruption in cholesterol trafficking to inner mitochondrial membrane, since StAR expression was inhibited. The results obtained also demonstrated that the expression of AGTR1, VEGFR2 and R3 were down-regulated following PGF 2α administration. Further, the data obtained also suggested modulation of expression of pro- and anti-angiogenic factors upon PGF2α-treatment indicative of an involvement of other autocrine or paracrine factor(s) in the regression of bovine CL. This was an interesting finding as it suggests a novel and potential functional relationship between angiogenesis and the luteolytic response of CL to PGF2α administration. In bovines, despite extensive research being carried out to examine factors involved in the regulation of development and function of the CL, the trophic factor(s) required for maintenance of CL function, especially, P4 biosynthesis and secretion are not well characterized. It was hypothesized that the function of the CL during its finite lifespan must be responsive to LH as well as to various growth factors. Thus, experiments were conducted to examine the effects of increased LH and GH/IGF-I on the maintenance of CL function during mid luteal phase and post PGF2α administration and the results of these studies are presented in Chapter IV. To elucidate the role of LH as a trophic factor in the regulation of CL function, effects of increased endogenous LH through GnRH administration and exogenous hCG injections were examined. The results indicated an absence of noticeable effect of various hCG/GnRH treatments on circulating P4 levels. On the other hand, administration of GH resulted in increased serum IGF-1 and P4 levels. It was further observed that the administration of a combination of hCG and GH increased serum P4 levels better than treatment with GH alone. Further experiments were carried out to examine the complex reciprocal relationship between LH/GH and PGF2α on expression of genes involved in the regulation of luteal structure and function. In buffalo cows, administration of exogenous hCG and/or GH following inhibition of CL function by PGF2α administration did not prevent the PGF2α-induced decline in serum P4 levels, but PGF2-mediated decrease in expression of LH/CGR and GHR genes was prevented upon GH administration. However, the decrease in StAR expression was not restored by hCG and GH treatments, thereby indicating that PGF2 action was not prevented by hCG and/or GH treatments. Taken together, the results of studies carried out in buffalo cows employing various experimental model systems suggest essential role for LH and GH/IGF-1, however, these factors were unable to reverse PGF2α-induced luteolysis. Further, our crucial findings of the effects of increased endogenous LH and IGF-1, in addition to their relationship with luteolytic agents such as PGF2α will open new avenues for studying the mechanisms involved in the regulation of structural and functional properties of the buffalo CL. It is well known that a large number of buffalo cows experience loss of pregnancy and infertility due to inadequate luteal function and/or failure of timely insemination. Results from our studies suggest that the incorporation of PGF2α and hCG or GH/IGF-1 protocols in buffalo cows to be beneficial for improving their breeding efficiency as these protocols are likely to increase luteal function with defined luteolysis. To summarize, the results of studies described in the present thesis provide new insights into the physiological and molecular mechanisms involved in the regulation of CL function during luteolysis in the monoovulatory species. The results suggest that the maintenance of CL function appears to be dependent on both luteotrophic and luteolytic factors, but with a varied degree of dominance between the two species examined. Further, the results indicate that while the luteotrophic factors (LH/CG) dominate the CL regulation in primates, the regulation of CL function in bovines is dominated by the actions of luteolytic factor (PGF2α). In monoovulatory species, the luteotrophic and luteolytic factors following binding to their specific plasma membrane receptors on the luteal cells, would counteract each other and modulate activation of various downstream signaling molecules subsequently leading to regulation of gene expression and P4 secretion (Fig.5.1). LH: luteinizing hormone; CG: chorionic gonadotropin; LH/CGR: LH/CG receptor; Gαs: stimulatory α-subunit of trimeric G-protein; AC: adenylate cyclase; cAMP: cyclic adenosine monophosphate; PKA: protein kinase A; p: phosphorylation: CREB: cAMP response element binding protein; SR-B1: scavenger receptor class B, type I; SF-1: steroidogenic factor 1; LRH-1: liver receptor homologue 1; P4; progesterone; Src; sarcoma; PDE4D: cAMP phosphodiesterase 4D; StAR, steroidogenic acute regulatory protein; PGF2α: prostaglandin F2α; PTGFR: PGF2α receptor; PLC: phospholipase C; CYP19A1: cytochrome P450 aromatase; PTGR1: Prostaglandin reductase 1; AREG: Amphiregulin; RTK: receptor tyrosine kinase; Akt: protein kinase B; FKHR: forkhead transcription factor; DAPL1: death associated protein like 1; ARG2: Arginase, type II Growth factor LH/CGR RR AC Gαs ? Gα TT P? Gα K PKP src cAMP ? P Akt PDE4D P PFKHR FKHR CREB P LRH-1CREB P SF-1 Genes associated with Genes associated with apoptosis ? CYP19A1, apoptosis SR-B1 PTGR1 DAPL1 SF-1, LRH-1 AREG ARG 2 P4 biosynthesis Apoptosis? P4 biosynthesis Apoptosis MONKEY BUFFALO COW Shown here is the diagram depicting intracellular signaling pathways regulated by luteotrophic factor (LH) and luteolytic factor (PGF2α) and their cross talk to counteract changes in the expressions of genes associated with the biosynthesis and secretion of P4 and apoptosis in the CL. In primates, LH/CG activates a multitude of intracellular signaling cascades, primarily Gαs/AC/cAMP/PKA/CREB leading to changes in gene expression. LH during early and mid luteal phase and CG during pregnancy maintain the activation of Src and PDE in an inhibitory state. However, during the late luteal phase of non-fertile cycle, results in present study suggests that activated Src levels and PDE activity increase, with accompanying decrease in cAMP and pCREB levels leading to concomitant decrease in SR-B1 expression, and in turn, P4 secretion. Surprisingly, regulation of apoptotic gene expression and CL regression are still unclear. In bovines, PGF2α of uterine origin mediates changes in luteal gene expression and results in decreased P4 secretion, principally by reduction in StAR level. The present study suggests that during luteolysis PGF2α affects the genes regulated by LH, by interfering with LH (and perhaps IGF-1) signaling leading to alteration in the expression of genes crucial for CL structure and function. (Pl refer the abstract file for figures)

Thesis (MSc (Biochemistry))--Stellenbosch University, 2008. / Gonadotropin-releasing hormone (GnRH) acting through the cognate GnRH receptor (GnRH-R) plays an important role in the regulation of mammalian reproductive function by regulating the synthesis and release of follicle stimulating hormone (FSH) and luteinizing hormone (LH). The sensitivity of pituitary gonadotropes to GnRH depends on the number of GnRH receptors present on the gonadotrope cell surface. GnRH-R is regulated at a transcriptional, post-transcriptional and post-translational level. Hormones such as GnRH and glucocorticoids (GCs) regulate GnRH-Rs in a time- and dose-dependent manner. Previous studies have shown that the GnRH-R promoter confers glucocorticoid-dependent activation via the activating protein 1 (AP-1) site in the nongonadotrope GGH3 cell line. The mechanism by which GCs regulate the GnRH-R promoter is not precisely known as the literature is contradictory. Therefore this study investigates the mechanism of transcriptional regulation of the mouse GnRH-R promoter in the mouse gonadotrope cell line LβT2, treated with the synthetic GC dexamethasone (dex). Assays used include promoter-reporter studies, Western blotting, endogenous mRNA expression studies, electrophoretic mobility shift assay (EMSA) as well as the in vivo chromatin immunoprecipitation (ChIP) assay. A transfected promoter-reporter plasmid containing 600 bp of the mouse GnRH-R promoter was used to investigate the effect of dex on transcriptional regulation. Previously it was determined in our laboratory that the GnRH-R promoter is activated via an AP-1 binding site in the LβT2 cell line, and is regulated in a time- and dose-dependent manner by dex. In the present study in the LβT2 cell line a small induction was indeed seen upon dex treatment. Cotransfecting a expression vector for rat GR succeeded in inducing a 2 fold positive dex response. Western blot analysis revealed that GR levels remain consistent even after 8 hours dex induction. The effect of dex on the endogenous GnRH-R gene was investigated by means of real-time RT-PCR. Dex did indeed upregulate the gene in a time-dependant manner. Maximal induction (7.4 fold) was obtained after at least 12 hours of dex treatment. Untreated LβT2 nuclear extracts were investigated using EMSA, for protein binding to the mouse GnRH-R promoter AP-1 binding site, and these proteins were identified as c- Fos and GR. This suggests that the GR interacts with the AP-1 transcription factor via a tethering mechanism to mediate the positive dex response. The results of an in vivo ChIP assay were consistent with this hypothesis, showing that the GR interacted with a genomic fragment containingthe AP-1 site, in response to dex. The transactivation of the GnRH-R promoter by means of the GR tethering to AP-1 has not been shown before in the LβT2 cell line.

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.

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.