Biomedical subjects

Susan Wray

Publications and source records attributed to Susan Wray.

At least 19 recordsLinked to original sources

PubMed2025

De novo rare EMX2 variants lead to idiopathic hypogonadotropic hypogonadism.

PURPOSE: The genetic etiology of infertility remains unknown. To identify genes for human infertility, we applied a de novo variant analysis in 142 parent-proband trios with idiopathic hypogonadotropic hypogonadism (IHH), an infertility disorder caused by gonadotropin-releasing hormone (GnRH) deficiency. METHODS: Rare de novo copy-number and single-nucleotide variants (CNVs and SNVs) were called from exome sequencing data of the IHH trios. An association study of common EMX2 variants and disease outcomes was performed in the Massachusetts General Brigham Biobank (N = 65,253). GnRH neuronal development and migration was studied in organotypic explants with knocked down of Emx2 and in a mouse model lacking Emx2. RESULTS: We identified that the gene EMX2 harbored both rare de novo CNVs and SNVs. Rare de novo EMX2 variants led to IHH, developmental delay, and hearing loss. Common EMX2 variants were linked to infertility, Parkinson disease, and hearing loss. Knockdown of Emx2 in nasal explants resulted in attenuated GnRH cell migration and GnRH cells were confined to nasal regions of Emx2 knockout (KO) mice, consistent with IHH pathogenesis. CONCLUSION: By utilizing a de novo variant analysis and cellular assays, EMX2 was uncovered as a gene for human infertility.

Humans↗

PubMed2025

Nongenomic Stimulatory Effect of T3 on Calcium Dynamics in GnRH Neurons via Integrin αVβ3.

Many clinical studies have identified correlations between thyroid dysfunction and reproductive issues, yet the underlying mechanisms behind this interaction remain poorly understood. In this study, we investigated the effect of triiodothyronine (T3) on the activity of gonadotropin-releasing hormone (GnRH) neurons, a key regulator of the central reproductive axis. Dual labeling confirmed that GnRH neurons express thyroid receptor (TR)α and integrin αVβ3 receptors mediating genomic and nongenomic effects of thyroid hormones, respectively. Using calcium imaging in an ex vivo model, we show that T3 induces a rapid and sustained increase of calcium oscillation frequency in GnRH neurons. No change in response was detected after application of T4. The T3 stimulatory effect was not inhibited by a TR-specific antagonist (1-850) but was mimicked by membrane-impermeable T3-BSA, indicating a mechanism independent of nuclear TR signaling. In contrast, the blockade of membrane αVβ3 integrins (with cilengitide) prevented the T3-induced increase in GnRH neurons calcium peak oscillation frequency. Further investigation using modulators of intracellular calcium and calcium entry revealed that binding to αVβ3 integrin can induce distinct calcium responses depending on the ligand, with T3 triggering a complex response involving multiple channels and calcium sources, possibly with compensatory mechanisms. In sum, these results demonstrate for the first time a direct effect of thyroid hormones on GnRH neuronal activity, with T3 stimulating calcium oscillations through the nongenomic αVβ3 integrin pathway. Understanding this thyroid-reproductive axis interaction will help clarify the mechanisms linking thyroid dysfunction to reproductive disorders and pave the way for targeted therapeutic interventions.

Animals↗

PubMed2007

Hepatocyte growth factor acts as a motogen and guidance signal for gonadotropin hormone-releasing hormone-1 neuronal migration.

Reproduction in mammals is under the control of the hypothalamic neuropeptide gonadotropin hormone-releasing hormone-1 (GnRH-1). GnRH-1-secreting neurons originate during embryonic development in the nasal placode and migrate into the forebrain along olfactory nerves. Gradients of secreted molecules may play a role in this migratory process. In this context, hepatocyte growth factor (HGF) is a potential candidate, because it promotes cell motility in developing brain and has been shown previously to act as a motogen on immortalized GnRH-1 neurons (GN11). In this study, the role of HGF and its receptor Met during development of the GnRH-1 system was examined. GnRH-1 cells express Met during their migration and downregulate its expression once they complete this process. Tissue-type plasminogen activator (tPA), a known HGF activator, is also detected in migratory GnRH-1 neurons. Consistent with in vivo expression, HGF is present in nasal explants, and GnRH-1 neurons express Met. HGF-neutralizing antibody was applied to explants to examine the role of the endogenous growth factor. Migration of GnRH-1 cells and olfactory axon outgrowth were significantly reduced, in line with disruption of a guidance gradient. Exogenous application of HGF to explants increased the distance that GnRH-1 cells migrated, suggesting that HGF also acts as a motogen to GnRH-1 neurons. Functional experiments, performed on organotypic slice cultures, show that creation of an opposing HGF gradient inhibits GnRH-1 neuronal migration. Finally, tPA(-/-):uPA(-/-) (urokinase-type plasminogen activator(-/-)) knock-out mice exhibit strong reduction of the GnRH-1 cell population. Together, these data indicate that HGF signaling via Met receptor influences the development of GnRH-1.

Animals↗

PubMed2006

Ontogeny of the GnRH systems in zebrafish brain: in situ hybridization and promoter-reporter expression analyses in intact animals.

The ontogeny of two gonadotropin-releasing-hormone (GnRH) systems, salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II), was investigated in zebrafish (Danio rerio). In situ hybridization (ISH) first detected sGnRH mRNA-expressing cells at 1 day post-fertilization (pf) anterior to the developing olfactory organs. Subsequently, cells were seen along the ventral olfactory organs and the olfactory bulbs, reaching the terminal nerve (TN) ganglion at 5-6 days pf. Some cells were detected passing posteriorly through the ventral telencephalon (10-25 days pf), and by 25-30 days pf, sGnRH cells were found in the hypothalamic/preoptic area. Continuous documentation in live zebrafish was achieved by a promoter-reporter expression system. The expression of enhanced green fluorescent protein (EGFP) driven by the sGnRH promoter allowed the earlier detection of cells and projections and the migration of sGnRH neurons. This expression system revealed that long leading processes, presumably axons, preceded the migration of the sGnRH neuron somata. cGnRH-II mRNA expressing cells were initially detected (1 day pf) by ISH analysis at lateral aspects of the midbrain and later on (starting at 5 days pf) at the midline of the midbrain tegmentum. Detection of red fluorescent protein (DsRed) driven by the cGnRH-II promoter confirmed the midbrain expression domain and identified specific hindbrain and forebrain cGnRH-II-cells that were not identified by ISH. The forebrain DsRed-expressing cells seemed to emerge from the same site as the sGnRH-EGFP-expressing cells, as revealed by co-injection of both constructs. These studies indicate that zebrafish TN and hypothalamic sGnRH cell populations share a common embryonic origin and migratory path, and that midbrain cGnRH-II cells originate within the midbrain.

Animals↗

PubMed2006

Cholecystokinin directly inhibits neuronal activity of primary gonadotropin-releasing hormone cells through cholecystokinin-1 receptor.

Pulsatile secretion of GnRH-1 regulates gonadotropin release from anterior pituitary and thus is essential for reproduction. The present study focused on the role of cholecystokinin (CCK) in the GnRH-1 system. CCK is a neuropeptide abundantly expressed in the brain, which is implicated in activation of female reproductive behaviors and release of anterior pituitary hormones. Using dual-label immunocytochemistry coupled to confocal analysis, GnRH-1 neurons in adult mouse brain were found to express CCK-1 receptors (CCK-1R), and CCK fibers were detected contacting GnRH-1 axons. To address the function of CCK on GnRH-1 neurons, calcium imaging was used to monitor patterns of activity of GnRH-1 neurons maintained in an in vitro system known to retain many characteristics of GnRH-1 cells in vivo. Endogenous receptors for CCK (CCK-1R and CCK-2R) were blocked with selective antagonists. Results indicate that CCK-1R but not CCK-2R antagonist treatment increased the number of calcium peaks/GnRH-1 cell, mean peak amplitude, and percentage of GnRH-1 cells displaying high activity. The increased activity in GnRH-1 neurons observed after application of CCK-1R antagonist was blocked by coincubation with exogenous CCK. This study provides evidence that CCK acts directly on GnRH-1 neurons to attenuate GnRH-1 neuronal activity via CCK-1R activation.

Age Factors↗

PubMed2006

Airway smooth muscle dysfunction precedes teratogenic congenital diaphragmatic hernia and may contribute to hypoplastic lung morphogenesis.

Fetal intervention aims to improve lung growth and survival in congenital diaphragmatic hernia (CDH). Airway smooth muscle (ASM) is important in lung development: ASM progenitors produce a key growth factor for lung morphogenesis (fibroblast growth factor 10); ASM contractility is also coupled to growth. ASM hyperreactivity occurs in postnatal CDH and may exacerbate barotrauma via impaired lung compliance. We hypothesize that ASM hyperreactivity and its sequelae are based on an early developmental lesion of ASM activity in hypoplastic lung. Sprague-Dawley rats were fed 100 mg nitrofen on Day 9.5 of pregnancy to induce lung hypoplasia in offspring (controls had vehicle alone). Normal and hypoplastic lung primordia were cultured from Day 13.5 of gestation at 37 degrees C in 5% CO(2) and loaded at 54 or 78 h with Ca(2+)-sensitive indicators: Fluo-4 for confocal imaging and Indo-1 or Fura-2 for photometric measurements of [Ca(2+)](i). Hypoplastic lung features spontaneous propagating ASM Ca(2+) transients with reduced frequency, increased amplitude, and significantly prolonged plateau duration, relative to control lung. Nonetheless, hypoplastic lung exhibits normal requirement for extracellular calcium entry and intracellular calcium release in initiation and regulation of ASM Ca(2+) waves. Early ASM dysfunction in lung hypoplasia is apparent as specific anomalies of Ca(2+) transients that indicate a problem with plasmalemmal ion channels/action potential generation. Elucidation of such an ASM lesion may allow pharmacologic amelioration not only of ASM hyperreactivity and its sequelae, but also of hypoplastic lung growth itself.

Animals↗

PubMed2005

Lipid rafts, the sarcoplasmic reticulum and uterine calcium signalling: an integrated approach.

The pathways involved in Ca2+ signalling in the uterus remain incompletely understood, impairing our ability to prevent preterm and difficult labours. In this review we focus on two elements in the pathway of Ca2+ signalling that have recently emerged as playing important roles: membrane lipid rafts and the sarcoplasmic reticulum. We examine the evidence for lipid rafts in the uterus and discuss their functional role. We suggest that the increases in cytosolic [Ca2+] and contractility that occur with raft disruption are due, at least in part, to effects on large conductance Ca2+-activated K+ (BK) channels that are localized to rafts. The role of the SR in contributing to subsarcolemmal cytosolic microdomains in uterus is evaluated, along with its interactions with ion channels on the plasma membrane. Thus, signalling microdomains play an important, but incompletely understood, role in the uterus, and integrating them into other Ca2+ signalling pathways is a challenge for further research. We suggest that the role of the SR changes in pregnancy, from promoting quiescence via BK channels or SR Ca2+ uptake, to promoting Ca2+ entry and contractility at term, and relate data on lipid rafts to clinical outcome in obese pregnant women.

Animals↗

PubMed2005

Action potential refractory period in ureter smooth muscle is set by Ca sparks and BK channels.

In excitable tissues the refractory period is a critical control mechanism preventing hyperactivity and undesirable tetani, by preventing subsequent stimuli eliciting action potentials and Ca2+ entry. In ureteric smooth muscle, peristaltic waves that occur as invading pacemaker potentials produce long-lasting action potentials (300-800 ms) and extraordinarily long (more than 10 s) refractory periods, which prevent urine reflux and kidney damage. For smooth muscles neither the mechanisms underlying the refractory period nor the link between excitability and refractoriness are properly understood. Here we show that a negative feedback process, which depends on Ca2+ loading the sarcoplasmic reticulum (SR) during the action potential and on the subsequent activation of local releases of Ca2+ from the SR (sparks), stimulating plasmalemmal Ca2+-sensitive K+ (BK) channels, determines the refractory period of the action potential. As sparks gradually reduce the Ca2+ load in the SR, electrical inhibition is released, the refractory period is terminated and peristaltic contractions occur again. The refractory period can be manipulated, for example from 10 s to 100 s, by altering the Ca2+ content of the SR or release mechanism or by inhibiting BK channels. This insight into the control of excitability and hence function provides a focus for therapies directed at pathologies of smooth muscle.

Action Potentials↗

PubMed2005

Spontaneous propagating calcium waves underpin airway peristalsis in embryonic rat lung.

Prenatal airways from diverse species exhibit spontaneous peristaltic contractions (airway peristalsis). These contractile waves appear coupled to and may function to regulate prenatal lung growth. They are unaffected by atropine or tetrodotoxin but abolished by nifedipine. Nevertheless, the mechanisms by which these contractile waves are generated, regulated, and propagated remain obscure. Using calcium imaging and whole embryonic lung organ culture, we demonstrate for the first time that peristalsis of the embryonic airway is driven by spontaneous, regenerative, temperature-sensitive calcium (Ca2+) waves. These Ca2+ waves propagate between individual airway smooth muscle cells coupled via gap junctions, are likely to be action potential-mediated, and are dependent on not only extracellular calcium entry via L-type voltage-gated channels but also intracellular Ca2+ stores. Thus, if airway peristalsis regulates lung growth, these findings mean that airway smooth muscle Ca2+ waves in turn regulate prenatal lung morphogenesis.

Animals↗

PubMed2005

Domain architecture of the smooth-muscle plasma membrane: regulation by annexins.

Individual signalling events are processed in distinct, spatially segregated domains of the plasma membrane. In a smooth muscle, the sarcolemma is divided into domains of focal adhesions alternating with caveolae-rich zones, both harbouring a specific subset of membrane-associated proteins. Recently, we have demonstrated that the sarcolemmal lipids are similarly segregated into domains of cholesterol-rich lipid rafts and glycerophospholipid-rich non-raft regions. In the present study, we provide a detailed structural analysis of the relationship between these proteinaceous and lipid domains. We demonstrate that the segregation of plasmalemmal protein constituents is intimately linked to that of the membrane lipids. Our results imply that lipid segregation is critical for the preservation of membrane protein architecture and essential for directional translocation of proteins to the sarcolemma. We show that the membrane lipid segregation is supported by the annexin protein family in a Ca2+-dependent manner. Eukaryotic cells harbour numerous, tissue-specific subsets of annexins. By examining the significance of this variety in a smooth muscle, we demonstrate that four different annexins target membrane sites of distinct lipid composition and that each annexin requires a different [Ca2+] for its translocation to the sarcolemma. Our results suggest that the interactions of annexins with distinct plasma membrane regions promote membrane segregation and, in combination with their individual Ca2+ sensitivity, might allow a spatially confined, graded response to a multitude of extra- or intracellular stimuli.

Annexins↗

PubMed2005

Modulation of agonist-induced Ca2+ release by SR Ca2+ load: direct SR and cytosolic Ca2+ measurements in rat uterine myocytes.

Release of Ca2+ from sarcoplasmic reticulum (SR) is one of the most important mechanisms of smooth muscle stimulation by a variety of physiologically active substances. Agonist-induced Ca2+ release is considered to be dependent on the Ca2+ content of the SR, although the mechanism underlying this dependence is unclear. In the present study, the effect of SR Ca2+ load on the amplitude of [Ca2+]i transients elicited by application of the purinergic agonist ATP was examined in uterine smooth muscle cells isolated from pregnant rats. Measurement of intraluminal Ca2+ level ([Ca2+]L) using a low affinity Ca indicator, mag-fluo-4, revealed that incubation of cells in a high-Ca2+ (10 mM) extracellular solution leads to a substantial increase in [Ca2+]L (SR overload). However, despite increased SR Ca2+ content this did not potentiate ATP-induced [Ca2+]i transients. Repetitive applications of ATP in the absence of extracellular Ca2+, as well as prolonged incubation in Ca2+-free solution without agonist, depleted the [Ca2+]L (SR overload). In contrast to overload, partial depletion of the SR substantially reduced the amplitude of Ca2+ release. ATP-induced [Ca2+]i transients were completely abolished when SR Ca2+ content was decreased below 80% of its normal value indicating a steep dependence of the IP3-mediated Ca2+ release on the Ca2+ load of the store. Our results suggest that in uterine smooth muscle cells decrease in the SR Ca2+ load below its normal resting level substantially reduces the IP3-mediated Ca2+ release, while Ca2+ overload of the SR has no impact on such release.

Adenosine Triphosphate↗

PubMed2005

Voltage-gated calcium channels in developing GnRH-1 neuronal system in the mouse.

Migration of gonadotropin-releasing hormone-1 (GnRH-1) neurons from the nasal placode into the central nervous system occurs in all vertebrates. This study characterizes the expression of L- and N-type voltage-gated calcium channels (VGCCs) in migrating GnRH-1 neurons in mice. Class C (L-type) and class B (N-type) VGCGs were detected in GnRH-1 cells and cells in the olfactory and vomeronasal epithelium during prenatal development. This expression pattern was mimicked in a nasal explant model known to retain many characteristics of GnRH-1 development in vivo. Using this in vitro system, perturbation studies were performed to elucidate the role of VGCCs in GnRH-1 neuronal development. This report shows that olfactory axon outgrowth and GnRH-1 neuronal migration are attenuated when nasal explants are grown in calcium-free media, and that this effect is temporally restricted to an early developmental period. Blockade of either the L- or the N-type channel did not alter GnRH-1 cell number or overall olfactory axon outgrowth. However, blockade of N-type channels altered the distribution of GnRH-1 neurons in the periphery of the nasal explants. In these explants, more GnRH-1 neurons were located proximal to, and fewer GnRH-1 neurons distal to, the main tissue mass, suggesting a general decrease in the rate of GnRH-1 neuronal migration. These results indicate that extracellular calcium is required for initiating GnRH-1 neuronal migration and that these events are partially dependent on N-type VGCC signals.

Animals↗

PubMed2004

Increased cholesterol decreases uterine activity: functional effects of cholesterol alteration in pregnant rat myometrium.

Uterine quiescence is essential for successful pregnancy. Cholesterol and triglycerides are markedly increased in pregnancy. Cholesterol is enriched in microdomains of the plasma membrane known as rafts and caveolae. Both lipid rafts and caveolae have been implicated in cellular signaling cascades. The purpose of this work was to investigate whether manipulation of cholesterol content alters uterine contractility. Late pregnancy (19-21 days) rats were humanely euthanized and strips of longitudinal myometrium were then dissected. Force and Ca(2+) measurements were simultaneously recorded and cholesterol increased by the addition of 5 mg/ml cholesterol or 0.25 mg/ml low-density lipoproteins (LDLs) or reduced by 2% methyl-beta-cyclodextrin (MCD) or 2 U/ml cholesterol oxidase addition to the perfusate. Both LDLs and cholesterol profoundly inhibited spontaneous uterine force production and associated Ca(2+) transients; frequency, amplitude, and duration of contraction were all significantly reduced compared with preceding control contractions. Force and Ca(2+) were also reduced by cholesterol when 1 nM oxytocin was used to stimulate the myometrium. Uterine activity was significantly increased by cholesterol extraction with MCD or cholesterol oxidase treatment. Electron microscopy confirmed the lipid raft disrupting effect of MCD, as formerly electron microscopy-visible caveolae in the myometrial cell membrane all but disappeared after MCD treatment. These data show that uterine smooth muscle cell cholesterol content is critically important for functional activity. A novel finding of our study is that cholesterol is inhibitory for force generation. It may be one of the mechanisms operating to maintain uterine quiescence throughout gestation and may also contribute to difficulties in labor suffered by obese women.

Animals↗

PubMed2004

Bovine serum albumin-estrogen compounds differentially alter gonadotropin-releasing hormone-1 neuronal activity.

Steroid hormones regulate a host of physiological processes and behaviors. These actions can occur by genomic mechanisms involving gene transcription or by nongenomic mechanisms proposed to involve receptors associated with the plasma membrane. BSA-conjugated steroid hormones have been extensively used to elucidate signal transduction pathways for these hormones. We have previously shown, using calcium imaging, that 17beta-estradiol (E2) significantly increases GnRH-1 neuronal activity. During the course of these experiments, it became apparent that three different BSA-estrogen compounds have been used in a variety of cell types: 17beta-estradiol 6-O-carboxymethyloxime-BSA (E2-6-BSA); 1,3,5(10)-estratrien-3,16alpha,17beta-triol-6-one 6-O-carboxymethyloxime-BSA (E-6-BSA); and 1,3,5(10)-estratrien-3,17beta-diol 17-hemisuccinate-BSA (E2-17-BSA). The effects of these compounds on GnRH-1 neuronal activity were compared using calcium imaging. E-6-BSA and E2-17-BSA, but not E2-6-BSA, significantly increased all parameters of GnRH-1 neuronal activity. In addition, the effects of these two BSA compounds were reversed by the estrogen receptor antagonist ICI 182,780 but not by inhibition of gene transcription. The effects of E2-17-BSA, but not E-6-BSA were reversed by treatment with pertussis toxin, which blocks G protein-coupled receptors. These data indicate that these compounds cannot be used interchangeably and clearly have different binding properties and/or different effects on target tissues.

Anesthetics, Local↗

PubMed2004

Vimentin-positive, c-kit-negative interstitial cells in human and rat uterus: a role in pacemaking?

The mechanism underlying spontaneous pacemaker potential in the uterus is not clearly understood. Several spontaneously active smooth muscles have interstitial cells of Cajal (ICCs) or ICC-like cells. We therefore examined cells from freshly dispersed uterine muscle strips (from pregnant human and rat myometrium) and in situ uterine preparations to determine the cell types present. Both preparations revealed numerous ICC-like cells; they were multipolar, with spider-like projections and enlarged central regions. These cells were readily distinguished from uterine myocytes by their morphology and ultrastructure, i.e., no myofilaments, numerous mitochondria, caveolae, and filaments. In addition, the ICC-like cells were noncontractile. These cells were negative to c-kit, a classic marker for ICCs. They stained positive for the intermediate filament, vimentin, a marker for cells of mesenchymal origin but not differentiated myocytes. The ICC-like cells had a more or less stable resting membrane potential of -58+/-7 mV compared with smooth-muscle cells, -65+/-13 mV, and produced outward current in response to voltage clamp pulses. However, in contrast with uterine myocytes, inward currents were not observed. This is the first description of ICC-like cells in myometrium and their role in the uterus is discussed, as possible inhibitors of intrinsic smooth-muscle activity.

Animals↗

PubMed2004

Rho-kinase inhibition and electromechanical coupling in rat and guinea-pig ureter smooth muscle: Ca2+-dependent and -independent mechanisms.

Recent data have shown Ca(2+)-dependent activation of Rho-kinase by sustained depolarization of arterial smooth muscle. Visceral smooth muscles, however, contract phasically in response to action potentials and it is unclear whether Ca(2+)-dependent or -independent Rho-kinase activation occurs. We have therefore investigated this, under physiologically relevant conditions, in intact ureter. Action potentials, ionic currents, Ca(2+) transients, myosin light chain (MLC) phosphorylation and phasic contraction evoked by action potentials in guinea-pig and rat ureter were investigated. In rat, but not guinea-pig ureter, three Rho-kinase inhibitors, Y-27632, HA-1077 and H-1152, significantly decreased phasic contractions and Ca(2+) transients. Voltage- and current-clamp data showed that Rho-kinase inhibition reduced the plateau component of the action potential, inhibited Ca(2+)-channels and, indirectly, Ca(2+)-activated Cl(-) channels. The Ca(2+) channel agonist Bay K8644 could reverse these effects. The K(+) channel blocker TEA could also reverse the inhibitory effect of Y-27632 on the action potential and Ca(2+) transient. Ca(2+) transients and inward current, activated by carbachol-induced sarcoplasmic reticulum Ca(2+)release, were not affected by Rho-kinase inhibition. Rho-kinase inhibition produced a Ca(2+)-independent increase in the relaxation rate of contraction, associated with acceleration of MLC dephosphorylation, which was sensitive to calyculin A. These data show for the first time that: (1) Rho-kinase has major effects on Ca(2+) signalling associated with the action potential, (2) this effect is species dependent and (3) Rho-kinase controls relaxation of phasic contraction of myogenic origin. Thus Rho-kinase can modulate phasic smooth muscle in the absence of agonist, and the mechanisms are both Ca(2+)-dependent, involving ion channels, and Ca(2+)-independent, involving MLC phosphorylation activity.

Action Potentials↗

PubMed2004

Direct action of estradiol on gonadotropin-releasing hormone-1 neuronal activity via a transcription-dependent mechanism.

Pulsatile secretion of gonadotropin-releasing hormone-1 (GnRH-1) is essential for reproduction. GnRH-1 induces gonadotropin release and is regulated by 17beta-estradiol (E2). Although a subpopulation of GnRH-1 neurons expresses estrogen receptor (ER) beta, it is unclear whether E2 acts directly on GnRH-1 neurons or indirectly through interneuronal connections. To test the hypothesis that E2 acts directly on GnRH-1 neurons to regulate neuronal activity, we used calcium imaging to monitor intracellular calcium oscillations in GnRH-1 neurons maintained in nasal explants. TTX was used to minimize synaptic input from other cells. Consistent with previous studies, TTX reduced the activity of individual GnRH-1 neurons to a basal level, while the population of cells maintained synchronized calcium oscillations. Exposure of GnRH-1 cells to TTX plus E2 increased the number of calcium peaks/cell, percentage of cells with > or =10 peaks, mean peak amplitude, and percentage of cells that contributed to each calcium pulse in explants maintained in vitro for 7 d (7 div) compared with TTX alone. These effects were induced within 30 min and were not mimicked by 17alpha-estradiol, E2 conjugated to BSA (which does not cross the plasma membrane), or seen at 21 div, when the percentage of GnRH-1 cells expressing ERbeta transcripts declines. In addition, these effects were inhibited by the ER antagonist ICI 182,780 and prevented by inhibition of gene transcription. These data suggest that, via ERbeta, E2 can rapidly act as a hormone-activated transcription complex and are the first to show that E2 directly increases GnRH-1 neuronal activity and synchronization.

Adenocarcinoma↗

PubMed2004

Gonadotropin releasing hormone-1 expression in incisors of mice.

GnRH-1 is a decapeptide hormone that regulates gonadal maturation and fertility. In brain, GnRH-1 is secreted by neurons residing mainly in the preoptic/hypothalamic area. These neurons arise from cells in the nasal placode during embryonic development. GnRH-1 mRNA and peptide in the nonhypothalamic region have been described, suggesting other functions of GnRH-1. This paper describes for the first time the expression of GnRH-1 in developing incisors in mice. At embryonic day (E) 12.5, GnRH-1 mRNA and peptide were localized in cells in oral and dental epithelia. At postnatal day (P) 6, before incisor eruption, GnRH-1 was expressed in cells in dental epithelial-derived structures that include the papillary layer, outer dental epithelium, stellate reticulum, stratum intermedium, and enamel-secreting ameloblast cell layer. GnRH-1 expression correlated with cell maturity, becoming stronger in cells farther away from the proliferative zone. From E12.5 through P6, GnRH-1 expression was not detected in neural crest-derived dental mesenchyme or in mesenchyme-derived structures that include dental papilla, dental follicle, and dentin-secreting odontoblast. In addition, GnRH-1 expression was not detected in molars, indicating that expression of GnRH-1 is differentially regulated in incisor vs. molars, with only the former exhibiting continuous growth in this species. In homozygous hypogonadal mice at P1, GnRH-1 peptide expression was not detected, yet incisors were present. However, morphological changes in cells between dental follicle and ameloblast cell layer were noted. Taken together, our results indicate that GnRH-1 expression, although not essential for initiation and formation of incisors, may be important in maturation and/or maintenance of these placodally derived structures.

Animals↗