bluuebunny/crossref_metadata_2025 huggingface dataset:
An integrated environmental perfusion chamber and heating system for long‐term, high resolution imaging of living cells
Authors: Hing W. A. , Poole C. A. , Jensen C. G. , Watson M.
Published: March 2003

This communication presents the design and application of an integrated environmental perfusion chamber and stage heating blanket suitable for time‐lapse video microscopy of living cells. The system consists of two independently regulated components: a perfusion chamber suitable for the maintenance of cell viability and the variable delivery of environmental factors, and a separate heating blanket to control the temperature of the microscope stage and limit thermal conduction from the perfusion chamber. Two contrasting experiments are presented to demonstrate the versatility of the system. One long‐term sequence illustrates the behaviour of cells exposed to ceramic fibres. The other shows the shrinking response of cultured articular cartilage chondrons under dynamic hyper‐osmotic conditions designed to simulate joint loading. The chamber is simple in design, economical to produce and permits long‐term examination of dynamic cellular behaviour while satisfying the fundamental requirements for the maintenance of environmental factors that influence cell viability.
Applications of microscopy to the deformation and fracture of rubber‐toughened polymers
Authors: Bucknall C. B.
Published: March 2003

The principal mechanisms of inelastic deformation in rubber‐toughened plastics, and in other similar multiphase polymers, are shear yielding and multiple crazing in the rigid matrix phase, and cavitation in the soft disperse phase. Microscopy has played a very important part in the identification and study of these mechanisms. However, no one technique has been predominant. Polarized‐light microscopy is useful for observing crazing, and has proved to be the most effective method for studying shear yielding, whereas transmission electron microscopy has provided valuable insights into the cavitation behaviour of complex rubber particles. Scanning electron microscopy has made significant contributions in the study of all three micromechanisms. In some microscopy investigations, thin sections have been strained in situ on the microscope stage. In others, specimens have been deformed in the bulk state before being polished and etched, or stained and microtomed, or coated with a thin layer of metal, in preparation for examination in the microscope. The importance of combining microscopy with other, complementary methods for studying deformation micromechanics is emphasized, and two novel methods for detecting cavitation in multiphase polymers are discussed briefly.
Visualizing filamentous actin on lipid bilayers by atomic force microscopy in solution
Authors: Shi D. , Somlyo A. V. , Somlyo A. P. , Shao Z.
Published: March 2003

The surface structure of actin filaments (F‐actin) was visualized at high resolution, by atomic force microscopy (AFM) in aqueous solution, in large paracrystals prepared on positively charged lipid monolayers. The increased stability of these closely packed specimens allowed us to show that both the long pitch (38 nm) and the monomer (5.8 nm) can be directly resolved by AFM in the contact mode. The right‐handed helical surface, distinguishable in high resolution images, was compared with reconstructed models based on electron microscopy. The height of the rafts, a measure of the actin filament diameter, was 10 ± 1 nm, whereas the smaller inter‐filament distance, 8 ± 1 nm, was consistent with interdigitation of the filaments. The 10 ± 1 nm F‐actin diameter is in good agreement with the results of fibre X‐ray diffraction. As such specimens are relatively easy to prepare without specialized equipment, this method may allow the study of the thin filaments in which F‐actin‐associated proteins are also present.
A nanometre‐scale non‐periodic structural variation in high temperature superconducting ceramics and the implications for properties
Authors: Barry J. C. , Alarco J. A.
Published: March 2003

Non‐periodic structural variation has been found in the high Tc cuprates, YBa2Cu3O7‐x and Hg0.67Pb0.33Ba2Ca2Cu3O8+δ, by image analysis of high resolution transmission electron microscope (HRTEM) images. We use two methods for analysis of the HRTEM images. The first method is a means for measuring the bending of lattice fringes at twin planes. The second method is a low‐pass filter technique which enhances information contained by diffuse‐scattered electrons and reveals what appears to be an interference effect between domains of differing lattice parameter in the top and bottom of the thin foil. We believe that these methods of image analysis could be usefully applied to the many thousands of HRTEM images that have been collected by other workers in the high temperature superconductor field. This work provides direct structural evidence for phase separation in high Tc cuprates, and gives support to recent stripes models that have been proposed to explain various angle resolved photoelectron spectroscopy and nuclear magnetic resonance data. We believe that the structural variation is a response to an opening of an electronic solubility gap where holes are not uniformly distributed in the material but are confined to metallic stripes. Optimum doping may occur as a consequence of the diffuse boundaries between stripes which arise from spinodal decomposition. Theoretical ideas about the high Tc cuprates which treat the cuprates as homogeneous may need to be modified in order to take account of this type of structural variation.
Transmission line probe based on a bow‐tie antenna
Authors: Oesterschulze E. , Georgiev G. , Müller‐Wiegand M. , Vollkopf A. , Rudow O.
Published: March 2003

A high transmission probe for scanning near‐field optical microscopy is discussed that is based on a bow‐tie antenna. The proposed design of the transmission line probe relies on batch‐fabricated hollow pyramidal silicon dioxide tips that are partly coated with aluminium to accomplish the tapered dipole antenna. Theoretical calculations of the field distribution were performed to investigate its optical properties. Results were compared with those of conventional aperture tips based on the same silicon dioxide tip configuration, and revealed unique properties with respect to the transmission efficiency.
Atomic force microscopy observations of in situ deformed materials: application to single crystals and thin films on substrates
Authors: Coupeau C. , Grilhé J.
Published: March 2003

An experimental apparatus which consists of a compression machine interfaced with an atomic force microscope has been realized and allows the in situ observation of a sample surface under compressive stress. Taking advantage of the high resolution offered by this microscopy, the equipment is particularly suited both to analysing the fine slip line structure of deformed single crystals, providing interesting complementary information about plastic mechanisms taking place in the bulk, and to characterizing the mechanical behaviour of thin films on substrates with the investigation of the buckling phenomenon.
Quantitative measurements in in situ straining experiments in transmission electron microscopy
Authors: Pettinari F. , Couret A. , Caillard D. , Molénat G. , Clément N. , Coujou A.
Published: March 2003

Several examples of recent studies by in situ straining experiments in a transmission electron microscope performed in the Toulouse group (France) are presented. In particular, quantitative measurements of the features of the dislocation motion are described. These examples deal with individual or collective propagation of dislocations, which are submitted to various types of obstacle. Different metallic materials are investigated: magnesium, intermetallics, aluminium alloys and γ phase of a superalloy.
A new sample preparation method for biological soft X‐ray microscopy: nitrogen‐based contrast and radiation tolerance properties of glycol methacrylate‐embedded and sectioned tissue
Authors: Loo Jr B. W. , Sauerwald I. M. , Hitchcock A. P. , Rothman S. S.
Published: March 2003

We describe the preparation of a biological tissue for imaging in a transmission soft X‐ray microscope. Sections of exocrine pancreas embedded in glycol methacrylate polymer, an embedding medium widely used in visible light and electron microscopy, were examined. Contrast was based primarily on the nitrogen content of the tissue, and dual‐wavelength imaging at the nitrogen K‐shell absorption edge was used to map the distribution and provide quantitative densitometry of both the protein and embedding matrix components of the sample. The measurements were calibrated by obtaining the absorption spectrum of protein near the nitrogen edge. The contrast was consistent and reproducible, making possible the first large‐scale X‐ray microscopic study on sections of plastic‐embedded soft tissue. At radiation doses of up to 108 Gray, much more than required for routine imaging, no distortion and little mass loss were observed. This sample preparation method should permit routine imaging of tissues in X‐ray microscopes, previously a difficult task, as well as multimodal imaging (using visible light, X‐ray, electron, and scanned probe microscopies) on the same sample.
Assessment of chloride secretion in human nasal epithelial cells by X‐ray microanalysis
Authors: Dragomir A. , Andersson C. , Åslund M. , Hjelte L. , Roomans G. M.
Published: March 2003

The genetic disease cystic fibrosis (CF) is due to defective epithelial chloride transport. Different treatments have been proposed that could restore chloride transport in CF patients. A new method is proposed for measuring the chloride secretion in easily accessible epithelial cells.

Fresh nasal epithelial cells were obtained by nasal brushing and made to attach to titanium grids for electron microscopy. Chloride efflux through the cystic fibrosis transmembrane regulator channel was stimulated by 20 µm forskolin and 100 µm isobutyl‐methylxanthine (IBMX), in standard Ringer's solution (SR). Chloride efflux through the calcium‐regulated channel was stimulated by 200 µm adenosine triphosphate (ATP) in SR. The cells were rinsed after the exposure, in order to remove the experimental medium, frozen and freeze‐dried. The elemental composition of the cells was determined by X‐ray microanalysis.

Rinsing with distilled water or ammonium acetate appeared to cause damage to the cells, whereas rinsing with isotonic mannitol preserved the ionic composition. Stimulation of cells from healthy controls with forskolin and IBMX in a chloride‐containing medium caused a significant (28 ± 6%) decrease in chloride concentration, which is indicative of net chloride efflux. In similar conditions, stimulation with ATP induced a 29 ± 5% decrease in the chloride concentration.

Stimulation of cells from CF patients with forskolin and IBMX in a chloride‐containing medium caused no significant change in the intracellular chloride concentration, whereas ATP stimulation induced a response similar to that obtained in cells from healthy controls.

It is concluded that X‐ray microanalysis of nasal epithelial cells may be used to determine chloride secretion in CF patients in an easily accessible cell type.
Generalized approach for accelerated maximum likelihood based image restoration applied to three‐dimensional fluorescence microscopy
Authors: Schaefer L. H. , Schuster D. , Herz H.
Published: March 2003

For deconvolution applications in three‐dimensional microscopy we derived and implemented a generic, accelerated maximum likelihood image restoration algorithm. A conjugate gradient iteration scheme was used considering either Gaussian or Poisson noise models. Poisson models are better suited to low intensity fluorescent image data; typically, they show smaller restoration errors and smoother results. For the regularization, we modified the standard Tikhonov method. However, the generic design of the algorithm allows for more regularization approaches. The Hessian matrix of the restoration functional was used to determine the step size. We compared restoration error and convergence behaviour between the classical line‐search and the Hessian matrix method. Under typical working conditions, the restoration error did not increase over that of the line‐search and the speed of convergence did not significantly decrease allowing for a twofold increase in processing speed. To determine the regularization parameter, we modified the generalized cross‐validation method. Tests that were done on both simulated and experimental fluorescence wide‐field data show reliable results.
Imaging the three orientation variants of the DO22 phase by 3D atom probe microscopy
Authors: Marteau L. , Pareige C. , Blavette D.
Published: March 2003

Three‐phase NiAlV alloys were investigated using a three‐dimensional atom probe. Ageing at 800 °C gives rise to the precipitation of two ordered phases within the supersaturated FCC solid solution, namely Ni3Al (L12 structure) and Ni3V (DO22 structure). The DO22 phase has three orientation variants which need to be identified in 3DAP images. It is shown that an appropriate choice of analysis site enables us to image the chemical order within both L12 and DO22 ordered phases and to distinguish the three orientation variants of the DO22 phase in reconstructed images. The lateral resolution of 3DAP in these experimental conditions was estimated through simple considerations to be less than 0.3 nm.
Evaluation of global analysis algorithms for single frequency fluorescence lifetime imaging microscopy data
Authors: Verveer P. J. , Bastiaens P. I. H.
Published: March 2003

Global analysis of fluorescence lifetime image microscopy (FLIM) data can be used to obtain an accurate fit of multi‐exponential fluorescence decays. In particular, it can be used to fit a bi‐exponential decay to single frequency FLIM data, which is not possible with conventional fitting techniques. Bi‐exponential fluorescence decay models can be used to analyse quantitatively single frequency FLIM data from samples that exhibit fluorescence resonance energy transfer (FRET). Global analysis algorithms simultaneously fit multiple measurements acquired under different experimental conditions to achieve higher accuracy. To demonstrate that bi‐exponential models can indeed be fitted to single frequency data, we derive an analytical solution for the special case of two measurements and use this solution to illustrate the properties of global analysis algorithms. We also derive a novel global analysis algorithm that is optimized for single frequency FLIM data, and demonstrate that it is superior to earlier algorithms in terms of computational requirements.
Labelling quality and chromosome morphology after low temperature FISH analysed by scanning far‐field and near‐field optical microscopy
Authors: Winkler R. , Perner B. , Rapp A. , Durm M. , Cremer C. , Greulich K.‐O. , Hausmann M.
Published: March 2003

A non‐enzymatic, low temperature fluorescence in situ hybridization (LTFISH) procedure was applied to metaphase spreads and interphase cell nuclei. In this context ‘low temperature’ means that the denaturation procedure of the chromosomal target DNA usually applied by heat treatment and chaotropic agents such as formamide was completely omitted so that the complete hybridization reaction took place at 37 °C. For LTFISH, the DNA probe had to be single‐stranded, which was achieved by means of separate thermal denaturation of the DNA probe only. The DNA probe pUC1.77 was used for all LTFISH experiments. The labelling quality (number of binding sites, relative background intensity, relative intensity of major and minor binding sites) was analysed by confocal laser scanning microscopy (CLSM). An optimum in specificity and signal quality was obtained for 15 h hybridization time. For this hybridization condition of LTFISH, the chromosomal morphology was analysed by scanning near‐field optical microscopy (SNOM). The results were compared with the morphology of chromosomes after (a) labelling of all centromeres using the same chemical treatment in the FISH procedure but with the application of target denaturation, and (b) labelling of all centromeres using a standard FISH protocol including thermal denaturation of the DNA probe and the chromosomal target. Depending on the FISH‐procedure applied, SNOM images show substantial differences in the chromosome morphology. After LTFISH the chromosome morphology appeared to be much better preserved than after standard FISH. In contrast, the application of the LTFISH chemical treatment accompanied by heat denaturation had a very destructive influence on chromosomal morphology. The results indicate that, at least for certain DNA probes, specific chromosome labelling can be obtained without the usually applied heat and chemical denaturation of the DNA target, resulting in an apparently well preserved chromatin morphology as visualized by SNOM. LTFISH may be therefore a useful labelling technique whenever the chromosomal morphology had to be preserved after specific labelling of DNA regions. Binding mechanisms of single‐stranded DNA probes to double‐stranded DNA targets are discussed.
Intratesticular Serotonin Affects Steroidogenesis in the Rat Testis
Authors: Csaba  , Csernus  , Gerendai
Published: March 2003

The effect of intratesticular administration of serotonin (5‐HT), ketanserin (5‐HT2 receptor antagonist), and 5,7‐dihydroxytryptamine (5,7‐DHT) (the neurotoxin that destroys serotoninergic neural elements) on steroidogenesis was studied in immature and adult rats. In adults, bilateral intratesticular injection of 5‐HT resulted in a significant decrease in basal but not in hCG‐stimulated testosterone secretion and in serum testosterone concentration, whereas ketanserin induced a significant rise in steroidogenesis 1 h post‐treatment. There was no effect 1 day after administration of 5‐HT or ketanserin, and 7 days after the injection of 5,7‐DHT. In immature rats 1 day after bilateral testicular administration of ketanserin, basal testosterone secretion in vitro was significantly suppressed. In immature hemicastrates, local injection of 5‐HT resulted (1 day post‐treatment) in a significant rise in steroidogenesis while administration of 5,7‐DHT decreased testosterone secretion 7 days after the injection of the neurotoxin. The results indicate that in adult rats 5‐HT exerts a suppressive, whereas in immature rats, a stimulatory action on steroidogenesis occurs. Data also suggest that, in both age groups, the effect of 5‐HT is mediated through 5‐HT2 receptors. The observation that in immatures administration of the neurotoxin resulted in an effect similar to that found following the treatment with the receptor antagonist suggests that, in this age group, 5‐HT derived from local neural elements might also be involved in the control of 5‐HT on Leydig cell steroidogenesis.
Frog chromaffin and Adrenocortical Cell Co‐Cultures: A Model for the Study of Medullary Control of Corticosteroidogenesis
Authors: Shepherd Stacie P. , Holzwarth Matilde A.
Published: March 2003

Phylogenetic, physiological and morphological evidence indicates that interactions between chromaffin and adrenocortical cells are involved in the differentiation and maintenance of function of both cell types. Chromaffin–adrenocortical interaction has become recognized as an important component of adrenocortical regulation; however, the mechanisms by which chromaffin cells modulate adrenocortical function are not well understood. To study directly chromaffin–adrenocortical cellular interactions, we developed primary frog (Rana pipiens) adrenal co‐cultures. In these co‐cultures, chromaffin cells extend processes that project towards or onto adrenocortical cells, mimicking their organization in vivo and indicating a potential for interaction between the two cell types. Cell survival and differentiation were optimized using a combination of NGF, FGF and histamine to enhance neurite outgrowth and fetal calf serum plus 10−10 M ACTH to maintain steroidogenesis. Characterization of the cells by immunocytochemistry and histochemistry showed that chromaffin cells maintain expression of catecholamine biosynthetic enzymes and that adrenocortical cells maintain expression of steroidogenic enzymes. Furthermore, chromaffin cells release catecholamines upon stimulation with carbamylcholine or potassium while adrenocortical cells sustain a basal secretion rate of aldosterone and corticosterone that is augmented 10–40‐fold by 0.1 nM to 10 nM ACTH. We therefore propose that these co‐cultures serve as a useful model system to study the cellular and molecular mechanisms by which chromaffin cells modulate adrenocortical cell function.
Blockade of Tyrosine Hydroxylase Activity in the Median Eminence Partially Reverses the Long Day‐Induced Inhibition of Pulsatile LH Secretion in the Ewe
Authors: Viguié Catherine , Picard Sophie , Thiéry Jean‐Claude , Malpaux Benoît
Published: March 2003

The photoperiod‐induced stimulation of LH secretion is associated with a decrease in dopamine content, as well as in the activity of its rate limiting enzyme, tyrosine hydroxylase (TH), in the median eminence (ME) of the ewe. We therefore hypothesize that ME‐TH activity can constitute a limiting factor of photoperiod‐induced inhibition of LH pulsatile secretion. To test this hypothesis, we studied whether the inhibition of ME‐TH activity can reverse the long day‐induced inhibition of LH. Using microdialysis, a 3 mM solution of alpha methyl‐p‐tyrosine (αMPT; a competitive inhibitor of TH), was administered in the ME of ovariectomized ewes bearing a 0.5 cm oestradiol implant at the beginning of a LD‐induced inhibition of LH secretion. The vehicle solution was infused for 4 h followed by a 3 mM αMPT solution infused for an additional 4 h. LH pulsatile secretory patterns within the same animal were compared between the control period and the αMPT period. αMPT infusion in the ME was associated with an increase in LH pulse frequency whereas it did not affect prolactin secretion. In conclusion, our results suggest that the inhibition of TH activity in the ME causes a stimulation of LH secretion in long‐day inhibited ewes.
N‐Methyl‐d‐Aspartic Acid Stimulation of Vasopressin Release: Role in Osmotic Regulation and Modulation by Gonadal Steriods
Authors: Swenson Kerry L. , Badre Shenon E. , Morsette Delmore J. , Sladek Celia D.
Published: March 2003

Previous experiments demonstrated that excitatory amino acids participate in the osmotic regulation of vasopressin secretion, but the specific involvement of N‐methyl‐
d‐aspartic acid (NMDA) receptors was not evaluated. This was demonstrated in the present studies. NMDA stimulated vasopressin release from perifused explants of the hypothalamo‐neurohypophyseal system (HNS), and osmotic stimulation of vasopressin release was inhibited by MK‐801 (10 μM) and AP5 (100 μM) NMDA receptor antagonists. The effective concentration of NMDA was dependent upon the Mg2+ concentration of the perifusate with stimulation observed at 1 μM NMDA in Mg2+‐replete compared with 5 μM in low‐Mg2+ medium. Previous experiments also demonstrated that estradiol and dihydrotestosterone (DHT) inhibited osmotically stimulated vasopressin secretion, and a nongenomic mechanism of action was suggested by the ability of steroids conjugated to bovine serum albumin to replicate the effect. Experiments were performed to explore the potential role of NMDA receptors in this mechanism. Estradiol (50 pg/ml) and DHT (3 ng/ml) inhibited NMDA stimulated vasopressin release in perifused HNS explants. These results suggest a role of NMDA receptors in the mediation of vasopressin secretion in osmotically stimulated release. Furthermore, estradiol and DHT may exert their inhibitory effect on osmotically stimulated vasopressin release via the NMDA receptor.
Role of Endogenous Opiates in Glucoprivic Inhibition of the Luteinizing Hormone Surge and Fos Expression by Preoptic Gonadotropin‐Releasing Hormone Neurones in Ovariectomized Steroid‐Primed Female Rats
Authors: Briski  , Sylvester
Published: March 2003

In female mammals, the preovulatory luteinizing hormone (LH) ‘surge’ elicits ovulation and the subsequent transformation of Graafian follicles into corpora lutea, and is thus a critical component of successful reproduction. In light of evidence that this surge is impaired as a consequence of caloric restriction, the following experiments utilized pharmacological strategies to determine whether glucose substrate homeostasis influences the magnitude and/or duration of this pivotal hormonal event. Groups of oestrogen‐and progesterone‐primed ovariectomized (OVX) rats were injected intravenously (i.v.) with the glucose antimetabolite, 2‐deoxy‐d‐glucose (2DG: 100 or 400 mg/kg), or the vehicle, saline, prior to onset of the expected LH surge. Other rats were pretreated with 2DG (100 μg/rat) or saline by an intracerebroventricular (i.c.v) route. While glucoprivation did not abolish the afternoon LH surge in these animals, mean plasma LH levels were significantly decreased in groups injected with the higher i.v. dose of 2DG or treated with this drug by an i.c.v route, relative to their vehicle‐injected controls. In other studies, i.c.v delivery of the opioid receptor antagonist, naltrexone (NALT), partially reversed the inhibitory effects of 2DG on the gonadal steroid‐induced LH surge. Dual‐label immunocytochemistry of tissue sections from the preoptic area and anterior hypothalamus of OVX, steroid‐primed rats revealed nuclear Fos‐immunoreactivity (‐ir) in a subpopulation of gonadotropin‐releasing hormone‐(GnRH‐)immunopositive neurones prior to maximal preovulatory LH release. Animals pretreated with 2DG i.c.v showed a significant decrease in mean numbers of GnRH neurones exhibiting Fos‐ir, whereas coadministration of 2DG and NALT resulted in numbers of double‐labelled neurones that were similar to those detected in the non‐drug‐treated controls. These studies show that magnitude of the LH surge is decreased by glucose substrate imbalance, and that regulatory effects of this metabolic challenge on the reproductive neuroendocrine axis is correlated with alterations in the transcriptional activation of preoptic GnRH neurones by gonadal steroid positive feedback. The present results also support a role for central opiatergic neurotransmission in glucoprivic regulation of cyclic LH secretion in this animal model.
Inhibition of Luteinizing Hormone Secretion and Expression of c‐fos and Corticotrophin‐Releasing Factor Genes in the Paraventricular Nucleus During Insulin‐Induced Hypoglycaemia in Sheep
Authors: Adam Clare L. , Findlay Patricia A.
Published: March 2003

Insulin can act within the brain to stimulate ovine luteinizing hormone (LH) secretion, but insulin‐induced hypoglycaemia inhibits LH via unknown brain sites, possibly involving corticotrophin‐releasing factor (CRF). Castrate male sheep, with (E+) or without (E−) subcutaneous oestradiol implants, were blood sampled every 12 min for 8 h. Insulin (0.25 or 0.5 IU/kg) was injected at 4 h via the carotid artery or jugular vein. All treatments reduced LH output with no differences between dose rate nor route of administration, but sensitivity was greater in E+ than E−sheep. There was no evidence for an effect of insulin on LH 0–1 h postinjection; however, 1–3 h after insulin, when hypoglycaemia was established, LH pulses were inhibited in both E+ and E− sheep (P<0.001). Additional intravenous (i.v.) glucose injections given 1 h (20 mmol) and 2 h (10 mmol) after insulin (0.5 IU/kg) were each followed by an LH pulse within 30 min (75% response in both E+ and E− sheep). In a separate experiment, sheep were killed 2 h after i.v. insulin (0.5 IU/kg) or saline. In‐situ hybridization revealed c‐fos mRNA in the paraventricular nucleus (PVN), but not in any other hypothalamic nuclei nor in the hindbrain; and this was linked with increased CRF gene expression in the PVN. Similar c‐fos and CRF gene expression was seen in insulin‐treated sheep given additional i.v. glucose (20 and 10 mmol, respectively, 40 and 20 min ante mortem), but not in saline‐treated controls. Therefore, insulin‐induced hypoglycaemia inhibited LH secretion, with oestradiol potentiating the effect, and was associated with gonadal steroid‐independent c‐fos gene expression and increased CRF gene expression in the PVN. The ovine PVN may be involved in mediating insulin‐induced hypoglycaemic inhibition of LH by a mechanism which might involve CRF.
Noradrenergic Neurotoxin Suppresses Gonadotropin‐Releasing Hormone (GnRH) and GnRH Receptor Gene Expression in Ovariectomized and Steroid‐Treated Rats
Authors: Kang  , Son  , Seong  , Choi  , Kwon  , Lee  , Kim
Published: March 2003

The present study was designed to investigate whether noradrenergic neurotransmission regulates the gene expression of gonadotropin‐releasing hormone (GnRH) in the preoptic area and GnRH receptor in the pituitary. To this end, N‐(2‐chloroethyl)‐N‐ethyl‐2‐bromobenzylamine (DSP4, 50 mg/kg), an intraperitoneal (i.p.) injection of selective noradrenergic neurotoxin, was administered 1 h before progesterone (1 mg) treatment in ovariectomized and estradiol‐treated prepubertal rats. Treatment with DSP4 effectively blocked the progesterone‐induced increase in hypothalamic noradrenaline content, but not dopamine content, indicating that DSP4 selectively inhibits noradrenergic neurotransmission. DSP4 significantly blocked progesterone‐induced increase in serum luteinizing hormone (LH) concentrations as well as GnRH release from hypothalamic fragments incubated in vitro. DSP4 concomitantly down‐regulated GnRH mRNA levels in the preoptic area, as determined by competitive reverse transcription‐polymerase chain reaction. DSP4 also clearly down‐regulated progesterone‐induced GnRH receptor mRNA levels in the pituitary, whereas it failed to alter LHβ mRNA levels. In summary, blockade of noradrenergic neurotransmission with DSP4 resulted in profound reductions of hypothalamic GnRH and pituitary GnRH receptor gene expression.
Characterization of TSH‐Positive Cells in Foetal Rat Pars Tuberalis that Fail to Express Pit‐1 Factor and Thyroid Hormone β2 Receptors
Authors: Sakai  , Sakamoto  , Ijima  , Matsubara  , Kato  , Inoue
Published: March 2003

The thyroid stimulating hormone (TSH)‐immunoreactive cells (TSH cells) in the pars tuberalis (PT‐TSH cell) of the male rat pituitary gland show an intense spot‐like TSH immunoreaction in the paranuclear cytoplasm. However, the ontogenic origin and characteristics of these spot‐like stained PT‐TSH cells remain to be elucidated. The present study was designed to investigate the distribution and characteristics of PT‐TSH cells in the foetal and adult rat pituitary by immunostaining for Pit‐1 factor and thyroid hormone receptors (TRs) and reverse transcriptase‐polymerase chain reaction (RT‐PCR). TSH cells first appeared in the PT at 15.5 days of gestation and were either stained diffusely throughout the cytoplasm or displayed a strongly stained, spotty appearance in the paranuclear region. By 15.5 days of gestation, the rostral part of the PT consisted of columnar epithelium, in which TSH immunoreactivity was spot‐like in the apical region of cytoplasm corresponding to the Golgi apparatus. At the 16.5 days of gestation, TSH cells were present in the pars distalis (PD); however, the cells were mostly larger and polygonal with strong staining throughout the cytoplasm. These differences between the PT and PD were retained throughout foetal and neonatal rat development. The TSH cells in the PD of the adult or gestational rat were observed to contain Pit‐1 factor by double immunostaining. However, TSH cells in the PT lacked Pit‐1 factor. RT‐PCR confirmed the absence of Pit‐1 and TRβ2 mRNA in the PT of the adult and late gestation rat pituitary gland. These results suggest that apparently distinct types of TSH cells in the PT develop independently from TSH cells in the PD.
Distribution and Seasonal Variations in Levels of Three Native GnRHs in the Brain and Pituitary of Perciform Fish
Authors: Senthilkumaran  , Okuzawa  , Gen  , Ookura  , Kagawa
Published: March 2003

Specific and sensitive radioimmunoassays (RIAs) were newly developed for two types of gonadotropin‐releasing hormone (GnRH), namely, seabream (sb) GnRH and chicken (c) GnRH‐II. We employed these two RIAs together with a previously reported RIA for salmon (s) GnRH to study the presence and regional distribution of these three GnRHs in the brains and pituitaries of four perciform fishes (red seabream, Pagrus major; black seabream, Acanthopagrus schlegeli; striped knifejaw, Oplegnathus fasciatus; and Nile tilapia, Oreochromis niloticus), as well as clarify seasonal changes in levels of these GnRHs in the brain and pituitary of red seabream. All three GnRHs were found in brains of all fishes examined, with regional distributions in the brains of the three GnRHs being rather similar. sbGnRH was abundant in telencephalon and hypothalamus. cGnRH‐II was concentrated from the middle to posterior part of the brain and distributed throughout the brain. sGnRH was concentrated in the olfactory bulb and distributed all over the brain, as was cGnRH‐II. The dominant form of GnRH in the pituitary was sbGnRH, with levels 500‐ to 2400‐fold higher than those of sGnRH, while cGnRH‐II was undetectable in all four species. In the brain and pituitary of female red seabream, levels of both brain and pituitary sbGnRH increased from October (immature phase) and reached a peak in April (spawning phase), reflecting the increase in gonadosomatic index and vitellogenesis. However, levels of sbGnRH remained high only in the pituitary of completely regressed fish in June. Levels of both sGnRH and cGnRH‐II in the brain were higher in the regressed phase and remained lower during the spawning phase. From these and previous results, it appears that sbGnRH is physiologically the most important form of GnRH in reproduction in red seabream and, probably, in other perciforms also.
Nitric Oxide Regulation of Gonadotrophin Secretion in Prepubertal Heifers1
Authors: Honaramooz  , Cook  , Beard  , Bartlewski  , Rawlings
Published: March 2003

Mechanisms responsible for the pulsatile release of gonadotrophin secretion in prepubertal heifers are not fully known. We have shown that an excitatory amino acid agonist, N‐Methyl‐D, l‐aspartic acid (NMA), induces an immediate release of luteinizing hormone (LH) and follicle stimulating hormone (FSH) in prepubertal heifers. Nitric oxide (NO) has also emerged as an important regulator of LH release in rats. This study was designed to test the role of NO in the regulation of gonadotrophin release as well as the possible mediation by NO of the effects of NMA and gonadotrophin releasing hormone (GnRH) on gonadotrophin secretion in heifer calves. In experiment 1, four groups of five prepubertal heifers (33 weeks old) received one of the following treatments: (1); N‐G‐nitro‐
l‐arginine methyl ester (
l‐NAME, a NO synthase inhibitor, 35 mg/kg, i.v., once); (2) NMA (4.7 mg/kg, i.v., once); (3)
l‐NAME+NMA (as above); and (4) Vehicle (saline, i.v.). All heifers in all groups were also challenged with a bolus injection of GnRH (10 ng/kg, i.v., once). Blood samples were collected every 15 min for 10 h.
l‐NAME was injected after the first blood sample, NMA after 2 h and GnRH after 6 h of blood sampling. Administration of
l‐NAME alone, suppressed the spontaneous pulses of LH (P<0.04). Heifers in the NMA group responded with a significantly greater LH release than did the heifers in the
l‐NAME+NMA group (P<0.05). Following the GnRH challenge, heifer calves treated with
l‐NAME or NMA had higher LH pulse responses than the controls (P<0.05). In a second experiment, four groups of five heifer calves (34 weeks old) were given one of the following treatments: (1)
l‐NAME (as above); (2)
l‐arginine, a NO precursor (ARG, 100 mg/kg/h, i.v. drip infused for 6 h starting 2 h after first blood sample was taken); (3)
l‐NAME+ARG (as above); and (4) Vehicle (saline i.v. bolus and drip for 6 h). Blood samples were taken every 10 min for 8 h. Administration of
l‐NAME suppressed the pulsatile release of LH and FSH (P<0.05). Compared to the control group, infusion of ARG by itself did not change the pattern of LH secretion (P>0.05); however, in heifers given
l‐NAME, ARG restored a normal pattern of LH pulses, similar to the control values (P>0.05). It was therefore concluded that NO is involved in the regulation of LH, and possibly FSH, secretion and that NO may mediate, at least in part, the stimulatory effects of NMA on LH, and to some extent FSH, release. The responses to GnRH led us to suggest that NO may have inhibitory effects on the pituitary and NMA may have increased pituitary sensitivity to GnRH.
Oestrogen Receptors in the Brainstem of the Female Sheep: Relationship to Noradrenergic Cells and Cells Projecting to the Medial Preoptic Area
Authors: Scott  , Rawson  , Pereira  , Clarke
Published: March 2003

Oestrogen regulates the secretion of gonadotropin releasing hormone (GnRH) and this could be mediated by noradrenergic systems originating in the brainstem. Whilst it is known that noradrenergic cells possess oestrogen receptors (ER), it is not known whether ER‐immunoreactive (‐ir) cells in the brainstem project to the regions of the hypothalamus in which GnRH neurons are found. We have used dual‐label immunocytochemistry to determine the extent to which ER‐α is found in noradrenergic cells in the brainstem of the ovariectomized (OVX) ewe. Noradrenergic/adrenergic cells were identified by immunostaining for dopamine β‐hydroxylase (DBH). Cells that stained for both DBH and ER were found in both the A1 and A2 cell groups, with the highest levels found in the most caudal regions. In the A1 group, at the most caudal extent, 73% of ER‐ir cells were DBH‐positive and 19% of DBH‐ir cells were ER‐positive. The degree of co‐localization decreased in a linear manner towards the rostral brainstem. In the caudal half of A2, 9–14% of ER‐ir cells were DBH‐positive and 20–25% of DBH cells were ER‐positive. Less than 2% of DBH‐ir cells in the A5 group were dual‐labelled and none of the cells in the A6 and A7 groups were ER‐positive. The retrograde tracer FluoroGold was injected into the preoptic area of nine OVX ewes and labelled cells were examined in the brainstem to determine the extent of co‐localization of ER. Only injections in the rostroventral part of the medial preoptic area near to the organum vasculosum of the lamina terminalis resulted in the labelling of cells in the brainstem. One ewe with very strong labelling of the brainstem was selected for detailed mapping. In the ventrolateral medulla, half the ER‐ir cells in the most caudal regions were retrogradely labelled. Almost all the ER‐ir cells in the mid‐region of the ventrolateral medulla were retrogradely labelled but no co‐localization of retrograde tracer and ER was observed rostral to obex. There were many ER‐ir cells and retrogradely‐labelled cells in the nucleus of the solitary tract but only a few double‐labelled cells. Similarly, numerous ER‐ir cells and retrogradely labelled cells were observed around the lateral edges of the caudal fourth ventricle and across to the lateral parabrachial nucleus but there were few double‐labelled cells. These results suggest differential regulation of noradrenergic cells by oestrogen, with a direct action of the hormone confined to the cells in the most caudal region of the A1 and A2 cell groups. The cells of the caudal ventrolateral medulla which contain ER‐ir cells that project to the preoptic area may be important in the mediation by noradrenaline of the actions of oestrogen on GnRH secretion in the ewe.
Anatomically Specific Changes in the Expression of Somatostatin, Growth Hormone‐Releasing Hormone and Growth Hormone Receptor mRNA in Diabetic Rats
Authors: Busiguina  , Argente  , García‐Segura  , Chowen
Published: March 2003

Growth hormone (GH) secretion is altered in poorly controlled diabetic animals. However, modifications in the hypothalamic neuropeptides that control GH secretion, somatostatin and GH‐releasing hormone (GHRH), as well as changes in the sensitivity of the hypothalamus and pituitary to the feedback effects of GH, are less clear. We have used RNase protection assays and in‐situ hybridization to address whether the mRNA expression of GH, somatostatin and GHRH, as well as of the GH receptor (GHR) in the hypothalamus and anterior pituitary, are altered in streptozotocin‐induced diabetic rats. After induction of diabetes, rats were treated with insulin twice daily for 3 weeks to obtain either poorly controlled (mean plasma glucose >300 mg/dl) or well‐controlled diabetic rats. Although no significant change in pituitary GH mRNA expression was found, the hypothalamic expression of GHRH and somatostatin mRNA was reduced in poorly‐controlled diabetic rats and returned to control values with normalisation of plasma glucose concentrations (P<0.0001 and P<0.002, respectively). Somatostatin mRNA expression was reduced only in the central portion of the periventricular nucleus, with no change being seen in the other areas of the periventricular nucleus or in the arcuate, suprachiasmatic or paraventricular nuclei. A significant decline in GHRH mRNA expression was observed in both the arcuate nucleus and ventromedial hypothalamus. Anterior pituitary GHR mRNA expression was significantly reduced in both well and poorly‐controlled diabetic rats, while there was no change in the hypothalamus. To examine whether the evolution time of the diabetes influences these parameters, in a subsequent experiment, diabetic rats received no insulin for 2 months. A significant decline in GHRH and somatostatin mRNA expression was also observed in these rats. In addition, pituitary GH mRNA expression declined significantly in long‐term diabetic rats. These results demonstrate that: (1) the expression of both GHRH and somatostatin declines specifically in anatomical areas involved in anterior pituitary hormone control; (2) GHR mRNA expression is decreased in the pituitary of diabetic rats, but not in the hypothalamus, and does not return to control values with normalisation of mean blood glucose concentrations; and (3) the evolution time of the diabetes is important for detecting some changes, including the decrease in pituitary GH mRNA expression.
The Pars Tuberalis: The Missing Link in the Photoperiodic Regulation of Prolactin Secretion?
Authors: Morgan
Published: March 2003

The endocrine function of the pars tuberalis of the pituitary gland has been an enigma for many years. Recent work suggests that one of its primary functions in seasonal mammals is to mediate photoperiodically regulated changes in prolactin secretion via an unidentified factor called tuberalin.
Guinea‐Pig Gonadotropin‐Releasing Hormone: Immunoreactivity and Biological Activity1
Authors: Gao  , Van den Saffele  , Giri  , Kaufman
Published: March 2003

The DNA sequence of the encoding gene predicts a unique structure for guinea‐pig gonadotropin‐releasing hormone (GnRH). We assessed the immunoreactivity of synthetic mammalian GnRH, of a synthetic peptide with predicted guinea‐pig GnRH structure, and of extracts from rat and guinea‐pig hypothalami, using two different RIA systems. Whereas immunoreactivity of mammalian and guinea‐pig GnRH was rather similar when using an antiserum with conformational specificity for mammalian GnRH (Root RR‐5 antiserum), binding of both peptides to an antiserum with sequential specificity (Kelch R‐13 antiserum) was markedly different. The findings for GnRH extracted from rat and guinea‐pig hypothalami were similar to those for the corresponding synthetic peptides. Assessment of in‐vivo biological activity of synthetic mammalian and guinea‐pig GnRH in the intact male guinea‐pig showed that both peptides stimulate LH secretion dose‐dependently, the response to mammalian GnRH being slightly greater at low dose. This study confirms that the GnRH expressed in the brain of the adult guinea‐pig differs from mammalian GnRH and indicates that mammalian and guinea‐pig GnRH display conformational similarity and that both can stimulate guinea‐pig luteinizing hormone secretion.
Two Olfactory Placode Derived Galanin Subpopulations: Luteinizing Hormone‐Releasing Hormone Neurones and Vomeronasal Cells
Authors: Key S. , Wray S.
Published: March 2003

In adult rodents, the peptide galanin is expressed in a subpopulation of hypothalamic luteinizing hormone‐releasing hormone (LHRH) neurones in an activity‐dependent manner. In this investigation, we examined whether galanin mRNA expression in mice was activated coincident with LHRH mRNA expression, as LHRH neurones differentiate from the olfactory placode. Using in situ hybridization, we show (i) that galanin mRNA is coexpressed in LHRH neurones prenatally, (ii) that there is a decrease in galanin mRNA expression relative to LHRH mRNA expression once LHRH mRNA positive/galanin mRNA positive neurones migrate out of the olfactory pit and into the nasal septum, and (iii) the presence of a novel population of galanin mRNA positive/LHRH mRNA negative expressing neurones in the olfactory pit/vomeronasal organ which do not migrate into the central nervous systenm (CNS). This study demonstrates that there are at least two populations of galanin mRNA expressing neurones arising from the olfactory placode; one that remains in nasal regions, is LHRH mRNA negative and whose function is unknown, and one which is coexpressed with LHRH. In addition, the temporal expression of galanin mRNA in LHRH cells indicates that initial activation and subsequent inactivation of galanin mRNA expression is independent of synaptic CNS connections.
The Distribution of Progesterone Receptor Immunoreactivity and mRNA in the Preoptic Area and Hypothalamus of the Ewe: Upregulation of Progesterone Receptor mRNA in the Mediobasal Hypothalamus by Oestrogen
Authors: Scott C. J. , Pereira A. M. , Rawson J. A. , Simmons D. M. , Rossmanith W. G. , Ing¶ N. H. , Clarke I. J.
Published: March 2003

The distribution of progesterone receptors (PR) was mapped in the hypothalamus of the ewe using immunocytochemistry. These results were confirmed using in situ hybridization with a sheep‐specific 35S‐labelled riboprobe. In addition, the effect of oestrogen on the level of PR mRNA in the hypothalamus was examined in ovariectomized (OVX) ewes following treatment with an oestrogen implant or without treatment. PR immunoreactive (‐ir) cells were readily detected in OVX animals. Labelled cells were observed in four main hypothalamic regions: the preoptic area (POA), including the organum vasculosum of the lamina terminalis, periventricular nucleus (PeVN), ventromedial nucleus (VMN) and the arcuate nucleus (ARC) (including the region ventral to the mamillary recess). In addition, lightly stained PR‐ir cells were observed in the supraoptic nucleus and a few PR‐ir cells were also found in the diagonal band of Broca. No PR‐ir cells were found in the brainstem. PR mRNA‐containing cells were found in the same hypothalamic regions as the PR‐ir cells. Image analysis of emulsion‐dipped slides following in situ hybridization indicated that oestrogen treatment increased (P<0.01) the mean number of silver grains/cell and the density of labelled cells in the VMN and ARC but had no effect on the level of PR mRNA expression in the POA or PeN. The distribution of PR‐containing cells in the hypothalamus is similar to that described in other species and all cells were located in nuclei that contain large populations of oestrogen receptor‐containing cells. These include regions implicated in the regulation of reproductive neuroendocrine function, and reproductive behaviour. Oestrogen and progesterone synergize to inhibit GnRH secretion and the present results suggest that these functions may involve cells of the VMN and ARC, with oestrogen acting to upregulate PR.