Browsing by Author "Millar, Robert P"
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- ItemOpen AccessAutocrine regulation of gonadotropin-releasing hormone in immortalized hypothalamic GT1-7 neurons(1994) Pithey, Anne Louise; Millar, Robert P; Dutlow, CliveThe existence of an ultrashort feedback mechanism regulating GnRH secretion has been supported from in vivo and in vitro studies. However, the complex synaptic connections of GnRH neurons with other neural elements made it difficult to determine whether the regulation was mediated by direct actions on the GnRH neurons or through actions on other interneurons. The recent development of the GnRH-secreting neuronal cell line, GT1, provided a model system for the study of neural regulation of a pure population of GnRH neurons. The present studies utilized GT1 -7 cells to investigate whether GnRH (at the level of the nerve terminal) influences the control of its own release. Preliminary studies determined the presence of GnRH mRNA in GT1-7 cells and established a cell culture system for the analysis of secretagogue-induced GnRH release. In this system GnRH release was shown to be spontaneous and was enhanced by the addition of K⁺, L-GLU, forskolin and PMA. Furthermore, K⁺- and forskolin-induced GnRH release was dependent on extracellular Ca²⁺. For the analysis of an ultrashort feedback mechanism, GT1-7 cells were cultured in 6-well plates to near confluence and then incubated in serum-free medium in the presence (1 nM- 1 μM) or absence of GnRH antagonist, Ant 27. Basal, K⁺-and forskolin-induced secretion of GnRH was monitored with antiserum 1076 which does not cross-react with Ant 27 at> 1 μM. Ant 27 treatment increased basal, K⁺- and forskolin-stimulated GnRH release in a dose-dependent manner. Total content was unaffected by 18 h treatment of GT1-7 cells with Ant 27. This suggests that the effects of Ant 27 are at the level of release and not biosynthesis. The presence of GnRH binding sites in the cells was demonstrated with ¹²⁵I-GnRH analog. These findings support the concept that GnRH, acting via autoreceptors, negatively controls its own release.
- ItemOpen AccessBrain distribution and release of Cholecystokinin octapeptide(1980) Hudson, Anne Mary; Millar, Robert PIn this thesis the in vitro release of immunoreactive CCK₈ (iCCK₈) from rat central nervous system preparations and the regulation of this release have been studied. Rat brain was dissected into the following regions; hypothalamus, cerebral cortex, striatum and thalamus, according to the method of Brownstein, Arimura, Sato et. al. (1975). CCK₈ was found to be distributed throughout these regions (range of 9 - 300 pmoles), with the highest concentration in cortex (300 pmol). In addition, low levels (range 9 - 30 pmoles) of CCK₈ were found in spinal cord, brain stem and cerebellum, in agreement with other workers. Immunohistochemical techniques have demonstrated CCK-like immunoreactivity in nerve cell bodies and fibres throughout brain, particularly in the cortex. Subcellular fractionation of rat brain was used to study the subcellular localisation of CCK. Tissue was homogenised to shear off nerve terminals (synaptosomes) which were purified and used to study the release of the peptide from hypothalamic and extrahypothalamic nerve endings.
- ItemOpen AccessCloning and characterisation of gonadotropin-releasing hormone receptors from species in non-mammalian vertebrate classes : amphibia and osteichthyes(1998) Troskie, Brigitte Elise; Illing, Nicola; Millar, Robert PTwo or more forms of gonadotropin-releasing hormone (GnRH) have been isolated from most vertebrate species. In most species, GnRH variants have been shown to occur in distinct areas of the peripheral and central nervous systems, the gonads and other peripheral organs. Although GnRH is a primary regulator of gonadotropin secretion, it has been shown to have additional roles such as the regulation of growth hormone secretion in goldfish and the inhibition of a potassium current (M-current) in amphibian sympathetic ganglia. This raises the possibility of the occurrence of multiple GnRH receptor subtypes. This thesis describes the cloning and characterisation of GnRH receptor subtypes from two nonmammalian vertebrates, the Amphibian, Xenopus laevis and the Osteichthyes, Carassius auratus (goldfish). Using degenerate primers designed to the mammalian GnRH receptors two putative receptor subtypes were identified from both X. laevis (X/a.1 and X/b.1) and goldfish (GfA and GfB) genomic DNA. The full-length cDNA for X/a.1, was cloned from pituitary cDNA. When transiently expressed in COS-1 cells, this clone showed a GnRH-dependent stimulation of inositol phosphates. No full-length clone for X/b.1 could be isolated using cDNA from several different tissues. A partially processed transcript was, however, amplified from sympathetic ganglia cDNA. These ganglia showed specific binding to a chicken GnRH II (cGnRH II) agonist and cGnRH II immunoreactivity was also detected in extracts from the ganglia. The expression, function and pharmacology of clone X/b.1, thus remains unknown, but the presence of cGnRH II-specific binding sites on membranes from the sympathetic ganglia with distinctly different pharmacology, implies the presence of a second GnRH receptor subtype in these neurons. Full-length cDNA clones of GfA and GfB were amplified from goldfish pituitary and brain cDNA respectively. These receptors had a 71% amino acid identity to each other and a 43% amino acid identity to the human GnRH receptor. The pharmacology of these two GnRH receptor subtypes was investigated by transient expression in COS-1 cells. The GfA and GfB receptors had different pharmacologies as demonstrated by their selectivities for GnRH analogues. In situ hybridisation revealed a distinct expression pattern of the goldfish GnRH receptor subtypes in the brain, gonads and liver (Dr R. Peter, University of Alberta). The full-length receptors cloned from the pituitaries and brain of X. /aevis and the goldfish have a low homology to the cloned mammalian GnRH receptors and have several different features, such as the presence of an intracellular carboxy-terminal tail. This thesis, describing the primary structure and characterisation of ligand selectivity of non-mammalian GnRH receptors, provides some useful foundations for future work towards understanding ligand recognition in the GnRH receptor. The description of multiple receptor subtypes in the goldfish and possibly in X. laevis also provides valuable information into alternative roles of GnRH and its receptor, which we are only beginning to understand.
- ItemOpen AccessCloning and charaterisation of the Thyrotrophin-releasing hormone receptor and Gonadotrophin-relasing hormone receptor from chicken pituitary gland(1998) Sun, Yuh-Man; Millar, Robert P; Illing, NicolaThe hypothalamic hormones, thyrotrophin-releasing hormone (TRH) and gonadotrophin-releasing hormone (GnRH), play pivotal roles in the growth and sexual maturation of chickens. In chickens, TRH regulates the release and synthesis of thyrotrophin (TSH) and also acts as a growth hormone-releasing factor. GnRH stimulates the release and synthesis of gonadotrophins (LH and FSH). TRH and GnRH are released and stored in the median eminence, and both hormones are transported into the pituitary gland via the hypophysial portal circulation. TRH and GnRH exert their physiological functions by binding to their specific receptors (TRH receptor and GnRH receptor, respectively) on the surface of cells in the pituitary gland. The activated receptors couple to guanine nucleotide-binding regulatory proteins (G proteins), Gq and/or G11, which in turn triggers the secondary messenger [1,2- diacylglycerol (DAG) and inositoltrisphosphate (IP3)] signalling cascade. The signalling generates the physiological effects of the hormones. The TRH-R and GnRH-R are members of G-protein coupled receptor (GPCR) family. The objective of this thesis was to clone and characterise the chicken TRH and GnRH receptors as useful tools for investigating the regulatory roles of TRH and GnRH receptors in the growth and sexual maturation of chickens. In addition, sequence information of the receptors would potentially assist in elucidating the binding sites and the molecular nature of the processes involved in receptor activation.
- ItemOpen AccessA comparison of regulatory mechanisms of luteinizing hormone prolactin and growth hormone exocytosis in permeabilized primary pituitary cells (Part 1) ; The effect of divalent cations on luteinizing hormone and prolactin exocytosis in permeabilized primary pituitary cells (Part 2)(1992) Franco, Sharone Elizabeth; Davidson, James S; Millar, Robert P
- ItemOpen AccessCongenital hypogonadotropic hypogonadism due to GnRH receptor mutations in three brothers reveal sites affecting conformation and coupling(Public Library of Science, 2012) Tello, Javier A; Newton, Claire L; Bouligand, Jerome; Guiochon-Mantel, Anne; Millar, Robert P; Young, JacquesCongenital hypogonadotropic hypogonadism (CHH) is characterized by low gonadotropins and failure to progress normally through puberty. Mutations in the gene encoding the GnRH receptor ( GNRHR1 ) result in CHH when present as compound heterozygous or homozygous inactivating mutations. This study identifies and characterizes the properties of two novel GNRHR1 mutations in a family in which three brothers display normosmic CHH while their sister was unaffected. Molecular analysis in the proband and the affected brothers revealed two novel non-synonymous missense GNRHR1 mutations, present in a compound heterozygous state, whereas their unaffected parents possessed only one inactivating mutation, demonstrating the autosomal recessive transmission in this kindred and excluding X-linked inheritance equivocally suggested by the initial pedigree analysis. The first mutation at c.845 C>G introduces an Arg substitution for the conserved Pro 282 in transmembrane domain (TMD) 6. The Pro282Arg mutant is unable to bind radiolabeled GnRH analogue. As this conserved residue is important in receptor conformation, it is likely that the mutation perturbs the binding pocket and affects trafficking to the cell surface. The second mutation at c.968 A>G introduces a Cys substitution for Tyr 323 in the functionally crucial N/DPxxY motif in TMD 7. The Tyr323Cys mutant has an increased GnRH binding affinity but reduced receptor expression at the plasma membrane and impaired G protein-coupling. Inositol phosphate accumulation assays demonstrated absent and impaired Gα q/11 signal transduction by Pro282Arg and Tyr323Cys mutants, respectively. Pretreatment with the membrane permeant GnRHR antagonist NBI-42902, which rescues cell surface expression of many GNRHR1 mutants, significantly increased the levels of radioligand binding and intracellular signaling of the Tyr323Cys mutant but not Pro282Arg. Immunocytochemistry confirmed that both mutants are present on the cell membrane albeit at low levels. Together these molecular deficiencies of the two novel GNRHR1 mutations lead to the CHH phenotype when present as a compound heterozygote.
- ItemOpen AccessDistribution and biosynthesis of LH-RH and structurally related peptides(1988) Dutlow, Clive M; Millar, Robert PBiologically active peptides are a class of molecules which can, in general, be grouped into families of structurally related moieties which have a wide somatic distribution and which subserve multiple physiological roles. Thus, the releasing factors of the hypothalamus or their structural analogues may carry out multiple endocrine, paracrine, or neurotransmitter functions outside the hypothalamus. This thesis describes the distribution and biosynthesis of Luteinizing Hormone-Releasing Hormone (LH-RH) and structurally related peptides in mammalian tissues and tumours. Two strategies were employed in this investigation: (a) Immunological screening of tissues for suitable sources of LH-RH and the partial characterization of the LH-RH immunoreactive peptides of selected tissues; (b) Complementary oligonucleotide screening of mRNA from certain tissues, cDNA and genomic libraries as well as genomic DNA restriction endonuclease fragments to establish the nature of the LH-RH precursor and related molecules. Acetic acid extracts of nineteen rat tissues were assayed in serial dilution for LH-RH immunoreactivity (LH-RH-IR). An antibody directed to the middle of the LH-RH sequence was used in these screening radioimmunoassays. LH-RH-IR was found in extrahypothalamic brain areas, in gastroenteropancreatic tissues, in the retina, the submandibular gland, the thyroid and the testes. In view of a putative role of endogeonous LH-RH-like immunoreactive peptides in intra-testicular regulation, the molecular nature of the LH-RH-like material detected in rat testes was investigated. Acetic acid extracts of adult rat testes were partially purified by LH-RH-immunoaffinity chromatography. On Sephadex G-100 this material separated into four major peaks of >100K, ~32K, ~5K and <4K daltons. The <4K peak of LH-RH-IR eluted differently on Sephadex G-25 and analytical reverse phase HPLC than did synthetic hypothalamic LH-RH decapeptide. Antibodies directed at the C-terminus of LH-RH gave higher estimates of LH-RH-IR for all the chromatographically separated testicular polypeptides than did N-terminally and middle directed antisera. The small testicular LH-RH-like peptides displaced radiolabelled LH-RH agonist from rat pituitary membranes more effectively than did the larger immunoreactive molecules. Monkey, pig and dog testes as well as dog Sertoli cell tumours were extracted with acetic acid and shown to contain LH-RH immunoreactive material similar to that of the rat. Human seminal plasma was also investigated as a source of LH-RH-IR. When pooled seminal plasma from azoospermic males was fractionated by gel filtration some fractions had significant amounts of the rat testicular type of LH-RH.
- ItemOpen AccessGnRH and neuropeptide regulation of gonadotropin secretion from cultured human pituitary cells(1988) Wormald, Patricia J; Millar, Robert PGonadotropin-releasing hormone (GnRH) and its superactive analogues are currently being used in the treatment of a number of endocrine disorders, such as endometriosis, precocious puberty, infertility and prostatic cancer. Selection of these analogues for clinical use have been previously based on their activities in animal models. This thesis has therefore investigated the binding characteristics of the human GnRH receptor, in comparison to those of the rat receptor, as well as the activities of a number of GnRH analogues for stimulating luteinising hormone (LH) and follicle stimulating hormone (FSH) secretion from cultured human pituitary cells. The establishment of a human pituitary bioassay system has further made possible the investigation of the direct regulatory roles of GnRH and other neuropeptides in man. To date, such studies in man have been performed in vivo and are thus complicated by the simultaneous interactions of numerous modulators.
- ItemOpen AccessGonadotropin releasing hormone receptor ligand interactions(1995) Flanagan, Colleen A; Millar, Robert PThe decapeptide, gonadotropin releasing hormone (GnRH), is the central regulator of reproductive function. It binds to receptors on the gonadotrope cells of the pituitary and stimulates release of luteinizing hormone (LH) and follicle stimulating hormone (FSH). Eleven different structural forms of GnRH have now been identified in various animal species. Chimaeric analogues of some of the variant forms of GnRH were synthesized in order to study the functional significance of the most common amino acid substitutions, which occur in positions 5, 7 and 8. Peptide binding affinities for sheep and rat GnRH receptors and potencies in stimulating LH and FSH release from cultured sheep pituitary cells and LH release from cultured chicken pituitary cells were measured. Histidine in position 5 decreased LH releasing potency in chicken cells, but slightly increased receptor binding affinity in rat and sheep membranes. Tryptophan in position 7 had minimal effect on GnRH activity in mammals, but increased LH release in chicken cells. Although differences in the structural requirements of mammalian and chicken GnRH receptors were anticipated, it was also found that rat GnRH receptors exhibited higher affinity for analogues with Tryptophan in position 7, than did sheep GnRH receptors. Substitutions in position 8 revealed the most marked differences in the structural requirements of mammalian and chicken GnRH receptors. Arginine was required for high GnRH activity in mammalian systems, but analogues with neutral substitutions in position 8 were more potent in chicken pituitary cells. The tolerance of position 8 substitutions, combined with the relatively small effects, in chicken cells, of incorporating a D-amino acid in position 6, indicate that the chicken GnRH receptor is less stringent than mammalian receptors in its recognition of peptide conformation. To examine how changes in ligand structure cause changes in receptor binding affinity and receptor activation, it was necessary to know the structures of the GnRH receptors. A protocol was developed for the purification of GnRH binding proteins from detergent-solubilized pituitary membranes, by affinity chromatography. This procedure yielded a protein which migrated as a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis, but was different from the recently cloned GnRH receptor. To test the proposal that the arginine residue in mammalian GnRH interacts with an acidic receptor residue, eight conserved acidic residues of the cloned mouse GnRH receptor were mutated to asparagine or glutamine. Mutant receptors were transiently expressed in COS-1 cells and tested for decreased preference for Arg⁸-containing ligands by ligand binding and inositol phosphate production. One mutant receptor, in which the glutamate residue in position 301 was mutated, exhibited decreased affinity for mammalian GnRH. The mutant receptor also exhibited decreased affinity for [Lys⁸]-GnRH, but unchanged affinity for [Gln⁸]-GnRH compared with the wildtype receptor, and increased affinity for the acidic analogue, [Glu⁸]-GnRH. This loss of affinity was specific for the residue in position 8, because the mutant receptor retained hiszh affinity for analogues with favourable substitutions in positions 5, 6 and 7. Thus, the Glu³⁰¹ residue of the GnRH receptor plays a role in receptor recognition of Arg⁸ in the ligand, consistent with an electrostatic interaction between these two residues. The Glu³⁰¹ and Arg⁸ residues were not required for the high affinity interactions of conformationally constrained peptides. This indicates that an interaction which involves these two residues may induce changes in the conformation of GnRH after it has bound to the receptor.
- ItemOpen AccessGPR54-dependent stimulation of luteinizing hormone secretion by neurokinin B in prepubertal rats(Public Library of Science, 2012) Grachev, Pasha; Li, Xiao Feng; Lin, Yuan Shao; Hu, Ming Han; Elsamani, Leena; Paterson, Stewart J; Millar, Robert P; Lightman, Stafford L; O'Byrne, Kevin TKisspeptin, neurokinin B (NKB) and dynorphin A (Dyn) are coexpressed within KNDy neurons that project from the hypothalamic arcuate nucleus (ARC) to GnRH neurons and numerous other hypothalamic targets. Each of the KNDy neuropeptides has been implicated in regulating pulsatile GnRH/LH secretion. In isolation, kisspeptin is generally known to stimulate, and Dyn to inhibit LH secretion. However, the NKB analog, senktide, has variously been reported to inhibit, stimulate or have no effect on LH secretion. In prepubertal mice, rats and monkeys, senktide stimulates LH secretion. Furthermore, in the monkey this effect is dependent on kisspeptin signaling through its receptor, GPR54. The present study tested the hypotheses that the stimulatory effects of NKB on LH secretion in intact rats are mediated by kisspeptin/GPR54 signaling and are independent of a Dyn tone. To test this, ovarian-intact prepubertal rats were subjected to frequent automated blood sampling before and after intracerebroventricular injections of KNDy neuropeptide analogs. Senktide robustly induced single LH pulses, while neither the GPR54 antagonist, Kp-234, nor the Dyn agonist and antagonist (U50488 and nor-BNI, respectively) had an effect on basal LH levels. However, Kp-234 potently blocked the senktide-induced LH pulses. Modulation of the Dyn tone by U50488 or nor-BNI did not affect the senktide-induced LH pulses. These data demonstrate that the stimulatory effect of NKB on LH secretion in intact female rats is dependent upon kisspeptin/GPR54 signaling, but not on Dyn signaling.
- ItemOpen AccessIdentification of human GnIH homologs, RFRP-1 and RFRP-3, and the cognate receptor, GPR147 in the human hypothalamic pituitary axis(Public Library of Science, 2009) Ubuka, Takayoshi; Morgan, Kevin; Pawson, Adam J; Osugi, Tomohiro; Chowdhury, Vishwajit S; Minakata, Hiroyuki; Tsutsui, Kazuyoshi; Millar, Robert P; Bentley, George EThe existence of a hypothalamic gonadotropin-inhibiting system has been elusive. A neuropeptide named gonadotropin-inhibitory hormone (GnIH, SIKPSAYLPLRF-NH 2 ) which directly inhibits gonadotropin synthesis and release from the pituitary was recently identified in quail hypothalamus. Here we identify GnIH homologs in the human hypothalamus and characterize their distribution and biological activity. GnIH homologs were isolated from the human hypothalamus by immunoaffinity purification, and then identified as MPHSFANLPLRF-NH 2 (human RFRP-1) and VPNLPQRF-NH 2 (human RFRP-3) by mass spectrometry. Immunocytochemistry revealed GnIH-immunoreactive neuronal cell bodies in the dorsomedial region of the hypothalamus with axonal projections to GnRH neurons in the preoptic area as well as to the median eminence. RT-PCR and subsequent DNA sequencing of the PCR products identified human GnIH receptor (GPR147) mRNA expression in the hypothalamus as well as in the pituitary. In situ hybridization further identified the expression of GPR147 mRNA in luteinizing hormone producing cells (gonadotropes). Human RFRP-3 has recently been shown to be a potent inhibitor of gonadotropin secretion in cultured sheep pituitary cells by inhibiting Ca 2+ mobilization. It also directly modulates GnRH neuron firing. The identification of two forms of GnIH (RFRP-1 and RFRP-3) in the human hypothalamus which targets human GnRH neurons and gonadotropes and potently inhibit gonadotropin in sheep models provides a new paradigm for the regulation of hypothalamic-pituitary-gonadal axis in man and a novel means for manipulating reproductive functions.
- ItemOpen AccessKisspeptin regulation of genes involved in cell invasion and angiogenesis in first trimester human trophoblast cells(Public Library of Science, 2014) Francis, Víctor A; Abera, Aron B; Matjila, Mushi; Millar, Robert P; Katz, Arieh AThe precise regulation of extravillous trophoblast invasion of the uterine wall is a key process in successful pregnancies. Kisspeptin (KP) has been shown to inhibit cancer cell metastasis and placental trophoblast cell migration. In this study primary cultures of first trimester human trophoblast cells have been utilized in order to study the regulation of invasion and angiogenesis-related genes by KP. Trophoblast cells were isolated from first trimester placenta and their identity was confirmed by immunostaining for cytokeratin-7. Real-time quantitative RT-PCR demonstrated that primary trophoblast cells express higher levels of GPR54 (KP receptor) and KP mRNA than the trophoblast cell line HTR8Svneo. Furthermore, trophoblast cells also expressed higher GPR54 and KP protein levels. Treating primary trophoblast cells with KP induced ERK1/2 phosphorylation, while co-treating the cells with a KP antagonist almost completely blocked the activation of ERK1/2 and demonstrated that KP through its cognate GPR54 receptor can activate ERK1/2 in trophoblast cells. KP reduced the migratory capability of trophoblast cells in a scratch-migration assay. Real-time quantitative RT-PCR demonstrated that KP treatment reduced the expression of matrix metalloproteinase 1, 2, 3, 7, 9, 10, 14 and VEGF-A, and increased the expression of tissue inhibitors of metalloproteinases 1 and 3. These results suggest that KP can inhibit first trimester trophoblast cells invasion via inhibition of cell migration and down regulation of the metalloproteinase system and VEGF-A.
- ItemOpen AccessKisspeptin signaling is required for the luteinizing hormone response in anestrous ewes following the introduction of males(Public Library of Science, 2013) De Bond, Julie-Ann P; Li, Qun; Millar, Robert P; Clarke, Iain J; Smith, Jeremy TThe introduction of a novel male stimulates the hypothalamic-pituitary-gonadal axis of female sheep during seasonal anestrus, leading to the resumption of follicle maturation and ovulation. How this pheromone cue activates pulsatile secretion of gonadotropin releasing hormone (GnRH)/luteinizing hormone (LH) is unknown. We hypothesised that pheromones activate kisspeptin neurons, the product of which is critical for the stimulation of GnRH neurons and fertility. During the non-breeding season, female sheep were exposed to novel males and blood samples collected for analysis of plasma LH profiles. Females without exposure to males served as controls. In addition, one hour before male exposure, a kisspeptin antagonist (P-271) or vehicle was infused into the lateral ventricle and continued for the entire period of male exposure. Introduction of a male led to elevated mean LH levels, due to increased LH pulse amplitude and pulse frequency in females, when compared to females not exposed to a male. Infusion of P-271 abolished this effect of male exposure. Brains were collected after the male effect stimulus and we observed an increase in the percentage of kisspeptin neurons co-expressing Fos, by immunohistochemistry. In addition, the per-cell expression of Kiss1 mRNA was increased in the rostral and mid (but not the caudal) arcuate nucleus (ARC) after male exposure in both aCSF and P-271 treated ewes, but the per-cell content of neurokinin B mRNA was decreased. There was also a generalized increase in Fos positive cells in the rostral and mid ARC as well as the ventromedial hypothalamus of females exposed to males. We conclude that introduction of male sheep to seasonally anestrous female sheep activates kisspeptin neurons and other cells in the hypothalamus, leading to increased GnRH/LH secretion.
- ItemOpen AccessKisspeptin signalling in the hypothalamic arcuate nucleus regulates GnRH pulse generator frequency in the rat(Public Library of Science, 2009) Li, Xiao-Feng; Kinsey-Jones, James S; Cheng, Yewsong; Knox, Alice M I; Lin, Yuanshao; Petrou, Nikoletta A; Roseweir, Antonia; Lightman, Stafford L; Milligan, Stuart R; Millar, Robert PBACKGROUND: Kisspeptin and its G protein-coupled receptor (GPR) 54 are essential for activation of the hypothalamo-pituitary-gonadal axis. In the rat, the kisspeptin neurons critical for gonadotropin secretion are located in the hypothalamic arcuate (ARC) and anteroventral periventricular (AVPV) nuclei. As the ARC is known to be the site of the gonadotropin-releasing hormone (GnRH) pulse generator we explored whether kisspeptin-GPR54 signalling in the ARC regulates GnRH pulses. METHODOLOGY/PRINCIPAL FINDINGS: We examined the effects of kisspeptin-10 or a selective kisspeptin antagonist administration intra-ARC or intra-medial preoptic area (mPOA), (which includes the AVPV), on pulsatile luteinizing hormone (LH) secretion in the rat. Ovariectomized rats with subcutaneous 17β-estradiol capsules were chronically implanted with bilateral intra-ARC or intra-mPOA cannulae, or intra-cerebroventricular (icv) cannulae and intravenous catheters. Blood samples were collected every 5 min for 5-8 h for LH measurement. After 2 h of control blood sampling, kisspeptin-10 or kisspeptin antagonist was administered via pre-implanted cannulae. Intranuclear administration of kisspeptin-10 resulted in a dose-dependent increase in circulating levels of LH lasting approximately 1 h, before recovering to a normal pulsatile pattern of circulating LH. Both icv and intra-ARC administration of kisspeptin antagonist suppressed LH pulse frequency profoundly. However, intra-mPOA administration of kisspeptin antagonist did not affect pulsatile LH secretion. Conclusions/Significance These data are the first to identify the arcuate nucleus as a key site for kisspeptin modulation of LH pulse frequency, supporting the notion that kisspeptin-GPR54 signalling in this region of the mediobasal hypothalamus is a critical neural component of the hypothalamic GnRH pulse generator.
- ItemOpen AccessQuantitative serial MRI of the treated fibroid uterus(Public Library of Science, 2014) Munro, Kirsty I; Thrippleton, Michael J; Williams, Alistair R W; McKillop, Graham; Walker, Jane; Horne, Andrew W; Newby, David E; Anderson, Richard A; Semple, Scott I; Marshall, Ian; Lewis, Steff C; Millar, Robert P; Bastin, Mark E; Critchley, Hilary O DObjective There are no long-term medical treatments for uterine fibroids, and non-invasive biomarkers are needed to evaluate novel therapeutic interventions. The aim of this study was to determine whether serial dynamic contrast-enhanced MRI (DCE-MRI) and magnetization transfer MRI (MT-MRI) are able to detect changes that accompany volume reduction in patients administered GnRH analogue drugs, a treatment which is known to reduce fibroid volume and perfusion. Our secondary aim was to determine whether rapid suppression of ovarian activity by combining GnRH agonist and antagonist therapies results in faster volume reduction. METHODS: Forty women were assessed for eligibility at gynaecology clinics in the region, of whom thirty premenopausal women scheduled for hysterectomy due to symptomatic fibroids were randomized to three groups, receiving (1) GnRH agonist (Goserelin), (2) GnRH agonist+GnRH antagonist (Goserelin and Cetrorelix) or (3) no treatment. Patients were monitored by serial structural, DCE-MRI and MT-MRI, as well as by ultrasound and serum oestradiol concentration measurements from enrolment to hysterectomy (approximately 3 months). RESULTS: A volumetric treatment effect assessed by structural MRI occurred by day 14 of treatment (9% median reduction versus 9% increase in untreated women; P = 0.022) and persisted throughout. Reduced fibroid perfusion and permeability assessed by DCE-MRI occurred later and was demonstrable by 2-3 months (43% median reduction versus 20% increase respectively; P = 0.0093). There was no apparent treatment effect by MT-MRI. Effective suppression of oestradiol was associated with early volume reduction at days 14 (P = 0.041) and 28 (P = 0.0061). CONCLUSION: DCE-MRI is sensitive to the vascular changes thought to accompany successful GnRH analogue treatment of uterine fibroids and should be considered for use in future mechanism/efficacy studies of proposed fibroid drug therapies. GnRH antagonist administration does not appear to accelerate volume reduction, though our data do support the role of oestradiol suppression in GnRH analogue treatment of fibroids. Trial Registration ClinicalTrials.gov NCT00746031
- ItemOpen AccessThe regulation of luteinizing hormone exocytosis in α-toxin permeabilized sheep anterior pituitary cells(1990) Van der Merwe, Philip Anton; Davidson, James S; Millar, Robert PAlthough exocytosis is the major mechanism by which cells secrete products into their environment, little is known about the mechanism of this fundamental process. Previous studies on the regulation of luteinizing hormone (LH) exocytosis have used intact cells exclusively. It is not possible, however, to determine the precise requirements for exocytosis in intact cells since the cytosol is not directly accessible. Permeabilization of the plasma membrane allows experimental manipulation of the intracellular milieu while preserving the exocytic apparatus. The diameter of the atoxin pores (2-3 nm) allowed the exchange of small molecules such as ATP while larger cytosolic proteins such as lactate dehydrogenase were retained. Because of the slow exchange of small molecules through a-toxin pores a protocol was developed which combines prolonged pre-equilibration of the permeabilized cells at 0°C before stimulation with strong Ca²⁺ buffering. Under these conditions an increase in the [Ca²⁺]free stimulated a 15-20 fold increase in LH exocytosis (EC₅₀ pCa 5.5). After 12-15 minutes the rate of exocytosis declined and the cells became refractory to Ca²⁺. At resting [Ca²⁺]free (pea 7), cAMP stimulated a rapid, 2 - 3 fold, increase in LH exocytosis. cAMP caused a modest enhancement of Ca²⁺-stimulated LH exocytosis by causing a left shift in the EC₅₀ for Ca²⁺ from pCa 5.6 to pCa 5.9. Activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA) synergistically enhanced cAMP-stimulated LH exocytosis, an effect which was further augmented by increasing the [Ca²⁺]free· Gonadotrophin-releasing hormone (GnRH) was found to stimulate cAMP production in intact pituitary cells. Since previous studies have shown that GnRH activates PKC and stimulates a rise in cytosolic [Ca²⁺]free, these results suggest that a synergistic interaction of the cAMP, PKC and Ca²⁺ second messenger systems is of importance in the mechanism of GnRH-stimulated LH exocytosis. When permeabilized cells were equilibrated for prolonged periods in the absence of MgATP, Ca²⁺-stimulated LH exocytosis declined. The time course of the decline closely followed the leakage of intracellular ¹⁴C-ATP. Addition of MgATP rapidly restored full Ca²⁺-stimulated LH exocytosis. Ca²⁺-, cAMP-, and PMA-stimulated LH exocytosis were all dependent on millimolar MgATP concentrations (EC₅₀ 1 .5-3 mM). It has been postulated that PKC is a mediator of Ca²⁺- stimulated exocytosis. Several findings in the present study argue against this hypothesis. Firstly, PMA and Ca²⁺ had additive effects on LH exocytosis at all [Ca²⁺]free· Secondly, PMA was able to stimulate further LH release from cells made refractory to high [Ca²⁺]free· Thirdly, the PKC inhibitor staurosporine did not inhibit Ca²⁺-stimulated LH exocytosis under conditions in which it inhibited PMAstimulated exocytosis. Fourthly, in cells desensitized to PMA by prolonged exposure to a high PMA concentrations, Ca²⁺-stimulated LH exocytosis was not inhibited. And finally, Ba²⁺+ was able to stimulate LH exocytosis to a maximal extent similar to Ca²⁺ despite the fact that Ba²⁺+ is an extremely poor activator of PKC. Since Ba²⁺+ is also a poor activator of calmodulin, this latter result implies that calmodulin does not mediate the effect of Ca²⁺. In agreement with this, the calmodulin inhibitor calmidazolium did not inhibit Ca²⁺-stimulated LH exocytosis. Since GTP-binding proteins have been implicated in regulated exocytosis in other cell systems, the effects of guanine nucleotides on LH exocytosis were examined. At resting cytosolic [Ca²⁺]free (pea 7), the GTP analogues GTPyS and GMPPNP stimulated LH exocytosis with similar potencies (EC₅₀ 20-50 μM). Additional experiments indicated that the effects of these GTP analogues could not be explained by activation of either PKC alone or cAMP-dependent protein kinase alone. In the presence of both PMA and cAMP, GMPPNP did not stimulate a further increase in the rate of LH exocytosis, suggesting that the stimulatory actions of guanine nucleotides may be mediated by the combined activation of PKC and generation of cAMP, as a result of activation of signal-transducing G proteins. In contrast, pretreatment of cells with GTPyS at low [Ca²⁺]free markedly inhibited subsequent responses to Ca²⁺, cAMP, PMA, and cAMP plus PMA. This inhibitory effect required lower GTPyS concentrations than the stimulatory effect (IC₅₀ 1-10 μM), and was not observed with GMPPNP. These findings indicate the involvement of a distinct guanine nucleotide-binding protein in exocytosis at a site distal to second messenger generation.
- ItemOpen AccessStructure and biological activity of avian hypothalamic luteinizing hormone-releasing hormone(1982) King, Judy A; Millar, Robert PIn 1971 Schally and co-workers (Schally et al., 1971) isolated gonadotropin-releasing hormone (now called luteinizing hormone-releasing hormone (LH-RH)) from sheep hypothalami and established that the hormone was a decapeptide with the amino acid sequence: pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH₂. The peptide was subsequently synthesised (Matsuo et al., 1971b) and shown to stimulate the release of gonadotropins (luteinizing hormone and follicle-stimulating hormone) in a wide range of mammalian species (Schally et al., 1973, 1976). With the exception of amphibians, nonmammalian vertebrates have a poor gonadotropin response to synthetic mammalian LH-RH (for reviews, see Ball, 1981; Jackson, 1981; King and Millar, 1981a). Since there is considerable molecular heterogeneity in the related neurohypophysial nonapeptide hormones (oxytocin-vasopressin) amongst vertebrates (Acher et al., 1972), we postulated that differences might exist in the structure of hypothalamic LH-RH in different vertebrate classes, Utilising a combination of regionspecific antisera and chromatographic techniques, we established that amphibian hypothalamic LH-RH is identical to the mammalian peptide while avian, reptilian, and piscine hypothalamic LH-RHs differ structurally in the region Gly⁶-Leu⁷-Arg⁸ (King and Millar, 1979a, 1980), We have now conducted further studies on avian hypothalamic LH-RH, which indicate that the arginine residue in position eight of mammalian LH-RH is substituted by glutamine in this vertebrate class. Purification of LH-RH from chicken hypothalami and determination of the amino acid composition have confirmed that the structure of avian LH-RH is: pGlu-His-Trp-Ser-Tyr-Gly-Leu-Gln-Pro-Gly-NH₂.