Shu Takigami

Alarm pheromone enhances stress-induced hyperthermia in rats

Behavioral and physiological effects of alarm pheromones emanating from stressed conspecific animals were investigated. Experimentally naive male Wistar rats were exposed to the test chambers containing alarm pheromones, which had been released by other rats receiving foot shocks in the same chamber beforehand. Along with behavioral analysis, the heart rate (HR) and core body temperature (cBT) were measured simultaneously using a biotelemetory system. Exposure to the alarm pheromones increased freezing, sniffing and walking and decreased resting as compared with rats exposed to control odors. In addition, these pheromone-exposed animals showed consistent increases in body temperature, i.e., stress-induced hyperthermia. After exposure to the alarm substances, immunoreactivity to nuclear Fos protein in the mitral cell layer in the accessory olfactory bulb (AOB) also increased compared with the reaction to control odors. These results suggest that an alarm pheromone enhances stress responses of conspecific animals both behaviorally and physiologically, and that these effects are mediated via activation of the AOB.

Microdissected Region-specific Gene Expression Analysis with Methacarn-fixed, Paraffin-embedded Tissues by Real-time RT-PCR

We have previously shown methacarn to be a versatile fixative for analysis of proteins, DNA, and RNA in paraffin-embedded tissues (PETs). In this study we analyzed its suitability for quantitative mRNA expression analysis of microdissected PET specimens using a real-time RT-PCR technique. Fidelity of expression in the methacarn-fixed PET sections, with reference to dose-dependent induction of cytochrome P450 2B1 in the phenobarbital-treated rat liver, was high in comparison with the unfixed frozen tissue case, even after hematoxylin staining. RNA yield from methacarn-fixed PET sections was equivalent to that in unfixed cryosections and was also not significantly affected by hematoxylin staining. Correlations between the expression levels of target genes and input amounts of extracted RNA in the range of 1–1000 pg were very high (correlation coefficients >0.98), the regression curves being similar to those with unfixed cryosections. Although cell numbers should be optimized for each target gene/tissue, ≥200 cells were necessary for accurate measurement in 10-μm-thick rat liver sections judging from the variation of measured value in small microdissected areas. These results indicate high performance with methacarn, close to that of unfixed tissues, regarding quantitative expression analysis of mRNAs in microdissected PET-specimens. (J Histochem Cytochem 52:903–913, 2004)

The expressed localization of rat putative pheromone receptors

The localization of pheromone receptors in the rat vomeronasal epithelium was examined by light- and electron-microscopic immunocytochemical analysis, using affinity-purified polyclonal antibodies. The antibodies were raised against a synthetic oligopeptide corresponding to a partial sequence of the rat putative pheromone receptor (VN6). Positive immunoreactivity was observed on the luminal surface of the sensory epithelium, and was abolished when an excess of the antigen peptide was added to the primary reaction solution. On electron microscopy, the immunoreactivity for the VN6 peptide was localized at the dendritic knobs and microvilli of receptor cells, but not in those of the supporting cells. These results show the first evidence of cellular localization of putative pheromone receptors in rat vomeronasal receptor cells.

Proton receptor GPR68 expression in dendritic-cell-like S100β-positive cells of rat anterior pituitary gland: GPR68 induces interleukin-6 gene expression in extracellular acidification

S100β-positive cells, which do not express the classical pituitary hormones, appear to possess multifunctional properties and are assumed to be heterogeneous in the anterior pituitary gland. The presence of several protein markers has shown that S100β-positive cells are composed of populations such as stem/progenitor cells, epithelial cells, astrocytes and dendritic cells. Recently, we succeeded in separating S100β-positive cells into round-cell (dendritic-cell-like) and process-cell types. We also found the characteristic expression of anti-inflammatory factors (interleukin-6, Il-6) and membrane receptors (integrin β-6) in the round type. Here, we further investigate the function of the subpopulation of S100β-positive cells. Since IL-6 is also a paracrine factor that regulates hormone producing-cells, we examine whether a correlation exists among extracellular acid stress, IL-6 and hormone production by using primary cultures of anterior pituitary cells. Dendritic-cell-like S100β-positive cells notably expressed Gpr68 (proton receptor) and Il-6. Furthermore, the expression of Il-6 and proopiomelanocortin (Pomc) was up-regulated by extracellular acidification. The functional role of IL-6 and GPR68 in the gene expression of Pomc during extracellular acidification was also examined. Small interfering RNA for Il-6 up-regulated Pomc expression and that for Gpr68 reversed the down-regulation of Il-6 and up-regulated Pomc expression by extracellular acidification. Thus, S100β-positive dendritic-like cells can sense an increase in extracellular protons via GPR68 and respond by the production of IL-6 in order to suppress the up-regulation of Pomc expression.

Expression of chemokine CXCL10 in dendritic-cell-like S100β-positive cells in rat anterior pituitary gland.

Chemokines are mostly small secreted polypeptides whose signals are mediated by seven trans-membrane G-protein-coupled receptors. Their functions include the control of leukocytes and the intercellular mediation of cell migration, proliferation, and adhesion in several tissues. We have previously revealed that the CXC chemokine ligand 12 (CXCL12) and its receptor 4 (CXCR4) are expressed in the anterior pituitary gland, and that the CXCL12/CXCR4 axis evokes the migration and interconnection of S100β-protein-positive cells (S100β-positive cells), which do not produce classical anterior pituitary hormones. However, little is known of the cells producing the other CXCLs and CXCRs or of their characteristics in the anterior pituitary. We therefore examined whether CXCLs and CXCRs occurred in the rat anterior pituitary lobe. We used reverse transcription plus the polymerase chain reaction to analyze the expression of Cxcl and Cxcr and identified the cells that expressed Cxcl by in situ hybridization. Transcripts of Cxcl10 and its receptor (Cxcr3 and toll-like receptor 4, Tlr4) were clearly detected: cells expressing Cxcl10 and Tlr4 were identified amongst S100β-positive cells and those expressing Cxcr3 amongst adrenocorticotropic hormone (ACTH)-producing cells. We also investigated Cxcl10 expression in subpopulations of S100β-positive cells. We separated cultured S100β-positive cells into the round-type (dendritic-cell-like) and process-type (astrocyte- or epithelial-cell-like) by their adherent activity to laminin, a component of the extracellular matrix; CXCL10 was expressed only in round-type S100β-positive cells. Thus, CXCL10 produced by a subpopulation of S100β-positive cells probably exerts an autocrine/paracrine effect on S100β-positive cells and ACTH-producing cells in the anterior lobe.

Expression of Slug in S100β-protein-positive cells of postnatal developing rat anterior pituitary gland

Among heterogeneous S100β-protein-positive (S100β-positive) cells, star-like cells with extended cytoplasmic processes, the so-called folliculo-stellate cells, envelop hormone-producing cells or interconnect homophilically in the anterior pituitary. S100β-positive cells are known, from immunohistochemistry, to emerge from postnatal day (P) 10 and to proliferate and migrate in the parenchyma of the anterior pituitary with growth. Recent establishment of S100β-GFP transgenic rats expressing specifically green fluorescent protein (GFP) under the control of the S100β-promoter has allowed us to observe living S100β-positive cells. In the present study, we first confirmed that living S100β-positive cells in tissue cultures of S100β-GFP rat pituitary at P5 were present prior to P10 by means of confocal laser microscopy and that they proliferated and extended their cytoplasmic processes. Second, we examined the expression of the Snail-family zinc-finger transcription factors, Snail and Slug, to investigate the mechanism behind the morphological changes and the proliferation of S100β-positive cells. Interestingly, we detected Slug expression in S100β-positive cells and its increase together with development in the anterior pituitary. To analyze downstream of SLUG in S100β-positive cells, we utilized specific small interfering RNA for Slug mRNAs and observed that the expression of matrix metalloprotease (Mmp) 9, Mmp14 and chemokine Cxcl12 was down-regulated and that morphological changes and proliferation were decreased. Thus, our findings suggest that S100β-positive cells express Slug and that its expression is important for subsequent migration and proliferation.

Degradation of serum microRNAs during transient storage of serum samples at 4

Background Numerous studies demonstrate the potential of circulating microRNAs as non-invasive biomarkers for several diseases. Circulating microRNAs are much more stable than mRNAs and remain largely intact even after prolonged incubation at room temperature. However, recent reports show that microRNAs in serum or plasma samples have diverse stabilities. The aim of this pilot study is to evaluate the stabilities of miR-92a, miR-122 and miR-145 in serum during transient storage at 4℃ before freezing. Methods Serum samples were stored for 24 h at 4℃, and then RNA was extracted from whole serum or extracellular vesicles in serum. Total Exosome Isolation Reagent (from serum) was used for the fractionation of extracellular vesicles. Reverse transcription and real-time PCR of microRNAs were performed using the TaqMan MicroRNA Assays for miR-92a, miR-122 and miR-145. Results MiR-122 and miR-145 were degraded rapidly in serum; the concentrations dropped to 35.9% (P < 0.001) and 29.3% (P < 0.0001), respectively. These microRNAs in extracellular vesicles exhibited similar instability; the concentrations were 52.2% (P < 0.05) and 56.5% (P < 0.01), respectively. On the other hand, no significant degradation of miR-92a was observed (whole serum: P = 0.052, extracellular vesicles: P = 0.196). Conclusions MiR-122 and miR-145 in serum are extremely unstable and could be degraded during transient storage of serum at 4℃ prior to freezing.

Identification of THY1 as a novel thyrotrope marker and THY1 antibody-mediated thyrotrope isolation in the rat anterior pituitary gland

Contact-dependent (juxtacrine) signaling is important for local cell-to-cell interaction and has received attention in recent years regarding its role in pituitary function, differentiation, and development. This study investigated one of the juxtacrine-related molecules, thymocyte differentiation antigen 1 (THY1), in the anterior lobe of the rat pituitary gland. Western blot analysis revealed expression of the THY1 protein in the adult rat anterior lobe. We also found that the THY1 ligand, integrin-β2 (ITGB2), is also expressed in the pituitary gland. In situ hybridization and immunohistochemical analyses showed that both THY1 mRNA and protein were present in almost, if not all, thyroid-stimulating hormone (TSH)-immunopositive cells (thyrotropes) and that ITGB2 was co-expressed in these cells. As THY1 appeared to represent a novel marker for thyrotropes, we then attempted to isolate these cells from various anterior lobe cells by the use of a THY1 antibody and a pluriBead-cascade cell isolation system. This technology allowed the isolation of thyrotropes with 83% purity at about 17-fold enrichment. Furthermore, the isolated THY1-immunopositive cells had higher Tsh mRNA levels compared with THY1-immunonegative cells and released TSH in response to thyrotropin-releasing hormone. These findings indicated that THY1 represents a potent thyrotrope marker and that the thyrotrope isolation method using the THY1 antibody may serve as a powerful tool to analyze their function including juxtacrine regulation through THY1/ITGB2 interaction.

Morphological assessment of the luminal surface of olfactory epithelium in mice deficient in tissue plasminogen activator following bulbectomy.

OBJECTIVE This study aimed to investigate the function of tissue plasminogen activator in the olfactory epithelium of mice following neural injury. METHOD Transmission electron microscopy was used to study the changes in the morphology of the olfactory epithelium 1-7 days after surgical ablation of the olfactory bulb (bulbectomy). RESULTS Prior to bulbectomy, a uniformly fine material was observed within some regions of the olfactory epithelium of mice deficient in tissue plasminogen activator. At 2-3 days after bulbectomy, there were degenerative changes in the olfactory epithelium. At 5-7 days after bulbectomy, we noted drastic differences in olfactory epithelium morphology between mice deficient in tissue plasminogen activator and wild-type mice (comparisons were made using findings from a previous study). The microvilli seemed to be normal and olfactory vesicles and receptor neuron dendrites were largely intact in the olfactory epithelium of mice deficient in tissue plasminogen activator. CONCLUSION The tissue plasminogen activator plasmin system may inhibit the regeneration of the olfactory epithelium in the early stages following neural injury.

Isolation and characterisation of CD9-positive pituitary adult stem/progenitor cells in rats

S100β protein and SOX2-double positive (S100β/SOX2-positive) cells have been suggested to be adult pituitary stem/progenitor cells exhibiting plasticity and multipotency. The aim of the present study was to isolate S100β/SOX2-positive cells from the adult anterior lobes of rats using a specific antibody against a novel membrane marker and to study their characteristics in vitro. We found that cluster of differentiation (CD) 9 is expressed in the majority of adult rat S100β/SOX2-positive cells, and we succeeded in isolating CD9-positive cells using an anti-CD9 antibody with a pluriBead-cascade cell isolation system. Cultivation of these cells showed their capacity to differentiate into endothelial cells via bone morphogenetic protein signalling. By using the anterior lobes of prolactinoma model rats, the localisation of CD9-positive cells was confirmed in the tumour-induced neovascularisation region. Thus, the present study provides novel insights into adult pituitary stem/progenitor cells involved in the vascularisation of the anterior lobe.