Three-dimensional cell culture using CD9-positive cells isolated from marginal cell layer of intermediate lobe of rats sustains in vivo-like primary niche environment.

The adenohypophysis is composed of the anterior and intermediate lobes (AL and IL, respectively), and secretes hormones that play an important role in reproduction. CD9- and SOX2-double (CD9/SOX2) positive cells located in the marginal cell layer (MCL) facing the Rathke's cleft in the AL and IL form the primary stem cell niche in the adult adenohypophysis of rats. In this study, we successfully obtained 3-dimensional (3D) cell aggregates that closely resembled the primary niche of MCL in vivo. After incubation in a Matrigel containing several growth factors, approximately 20% of the cells in the CD9/SOX2-positive cell aggregates were differentiated into hormone-producing cells. The cell aggregates generated in this study may provide insight into the regulation of the pituitary stem/progenitor cell niche and the turnover of hormone-producing cells.

Fluctuation of CD9/SOX2-positive cell populations during the turnover of GH- and TSH-producing cells in the adult anterior pituitary gland.

The adenohypophysis is comprised of the anterior and intermediate lobes (AL and IL, respectively). Cluster of differentiation 9 (CD9)- and sex-determining region Y-box 2 (SOX2)-positive cells are stem/progenitor hormone-producing cells in the AL. They are located in the marginal cell layer (MCL) facing Rathke's cleft between the AL and IL (primary niche) and the parenchyma of the AL (secondary niche). We previously showed that, in rats, CD9/SOX2-positive cells in the IL side of the MCL (IL-side MCL) migrate to the AL side (AL-side MCL) and differentiate into prolactin-producing cells (PRL cells) in the AL parenchyma during pregnancy, lactation, and diethylstilbestrol treatment, all of which increase PRL cell turnover. This study examined the changes in CD9/SOX2-positive stem/progenitor cell niches and their proportions by manipulating the turnover of growth hormone (GH)- and thyroid-stimulating hormone (TSH)-producing cells (GH and TSH cells, respectively), which are Pit1 lineage cells, as well as PRL cells. After induction, the isolated CD9/SOX2-positive cells from the IL-side MCL formed spheres and differentiated into GH and TSH cells. We also observed an increased GH cell proportion upon treatment with GH-releasing hormone and recovery from continuous stress and an increased TSH cell proportion upon propylthiouracil treatment, concomitant with alterations in the proportion of CD9/SOX2-positive cells in the primary and secondary niches. These findings suggest that CD9/SOX2-positive cells have the potential to supply GH and TSH when an increase in GH and TSH cell populations is required in the adult pituitary gland.

The multiciliated cells in Rathke's cleft express CYP26A1 and respond to retinoic acid in the pituitary.

The adenohypophysis consists of the anterior and intermediate lobes (AL and IL). The marginal cell layer (MCL), including the ventral region of the IL and the dorsal region of the AL lining the Rathke's cleft, acts as the primary stem/progenitor cell niches in adult adenohypophysis. The cells of the MCL on the IL side consisted of cluster of differentiation 9 (CD9)-positive stem/progenitor cells with or without motile cilia. However, any additional cellular properties of multiciliated CD9-positive cells are not known. The present study aimed to identify the character of the multiciliated cells in stem cell niche of the pituitary gland. We observed the fine structure of the multiciliated cells in the MCL of male Wistar rats at an early stage after birth and in adulthood (P60) using scanning electron microscopy. Since the previous study showed that the MCL cells of adult rats synthesize retinoic acid (RA), the present study determined whether the multiciliated cells are involved in RA regulation by the expression of retinal aldehyde dehydrogenase 1 (RALDH1) and CYP26A1, an enzyme synthesizing and degrading RA, respectively. Results showed that 96% of multiciliated cells in adult male rats expressed CYP26A1, while 60% expressed RALDH1. Furthermore, the isolated CD9-positive cells from the IL side MCL responded to RA and activated the degradation system of RA by increasing Cyp26a1 expression. These findings indicated that multiciliated cells are involved in RA metabolism in the MCL. Our observations provide novel insights regarding the stem cell niche of the adult pituitary.

Differentiation of stem progenitor CD9/SOX2-positive cells is promoted with increased prolactin-producing and endothelial cells in the pituitary.

Sex-determining region Y-box 2 (SOX2)-positive cells are stem/progenitor cells in the adenohypophysis, comprising the anterior and intermediate lobes (AL and IL, respectively). The cells are located in the marginal cell layer (MCL) facing Rathke's cleft (primary niche) and the parenchyma of the AL (secondary niche). We previously demonstrated in vitro that the tetraspanin superfamily CD9 and SOX2 double-positive (CD9/SOX2-positive) cells in the IL-side MCL migrate to the AL side and differentiate into hormone-producing and endothelial cells in the AL parenchyma. Here, we performed in vivo studies to evaluate the role of IL-side CD9/SOX2-positive cells in pregnancy, lactation, and treatment with diethylstilbestrol (DES; an estrogen analog) when an increased population of prolactin (PRL) cells was observed in the AL of the rat pituitary. The proportions of CD9/SOX2-, CD9/Ki67-, and PRL/TUNEL-positive cells decreased in the primary and secondary niches during pregnancy and DES treatment. In contrast, the number of CD9/PRL-positive cells increased in the AL-side MCL and AL parenchyma during pregnancy and during DES treatment. The proportion of PRL/Ki67-positive cells increased in the AL-side MCL and AL parenchyma in response to DES treatment. Next, we isolated CD9-positive cells from the IL-side MCL using an anti-CD9 antibody. During cell culture, the cells formed free-floating three-dimensional clusters (pituispheres). Furthermore, CD9-positive cells in the pituisphere differentiated into PRL cells, and their differentiation potential was promoted by DES. These findings suggest that CD9/SOX2-positive cells in the IL-side MCL may act as adult stem cells in the AL parenchyma that supply PRL cells under the influence of estrogen.

Cluster of differentiation (CD) 9-positive mouse pituitary cells are adult stem/progenitor cells.

SOX2-positive cells are stem/progenitor cells that supply hormone-producing cells; they are found in the anterior lobe of the rodent pituitary gland. However, they are likely composed of several subpopulations. In rats, a SOX2-positive cell populations can be distinguished by the presence of S100ß. We identified the novel markers cluster of differentiation (CD) CD9 and CD81, members of the tetraspanin superfamily, for the identification of S100ß/SOX2-positive cells. Recently, CD9/CD81 double-knockout mice were generated. Although they grew normally until 3 weeks after birth, they exhibited atrophy of the pituitary gland. These findings suggested that CD9/CD81/S100ß/SOX2-positive cells in the mouse pituitary are adult stem/progenitor cells. To substantiate this hypothesis, we examined CD9 and CD81 expression in the adult and developing anterior lobe. Immunohistochemistry showed that CD9/CD81-positive cells began appearing from postnatal day 0 and settled in the stem cell niches (marginal cell layer and parenchyma) of the adult anterior lobe while expressing S100ß. We next isolated CD9 -positive cells from the adult anterior lobe, using the anti-CD9 antibody for cell characterisation. The cells in culture formed free-floating three-dimensional clusters (pituispheres); moreover, induction into all types of hormone-producing cells was successful. Furthermore, reduction of CD9 and CD81 mRNAs by siRNAs inhibited cell proliferation. These findings indicate that CD9/CD81/S100ß/SOX2-positive cells may play a role as adult stem/progenitor cells in SOX2-positive subpopulations, thus supplying hormone-producing cells in the postnatal anterior lobe. Furthermore, CD9 and CD81 are implicated in cell proliferation. The current findings provide novel insights into adult pituitary stem/progenitor cells.

CD9-positive cells in the intermediate lobe of the pituitary gland are important supplier for prolactin-producing cells in the anterior lobe.

A supply of hormone-producing cells from stem/progenitor cells is critical to sustain the endocrine activity of the pituitary gland. In the adenohypophysis composing the anterior and intermediate lobe (AL and IL, respectively), stem/progenitor cells expressing sex-determining region Y-box 2 (SOX2) and S100ß are located in the marginal cell layer (MCL) facing Rathke's cleft (primary niche) and the parenchyma of the AL (secondary niche). Our previous studies using mice and rats indicated that the tetraspanin superfamily CD9 and CD81 are expressed in S100ß/SOX2-positive cells of primary and secondary niches (named CD9/CD81/S100ß/SOX2-positive cell), and the cells located in the AL-side niches exhibit plasticity and multipotency. However, it is unclear whether CD9/CD81/S100ß/SOX2-positive cells in the IL-side primary niche are stem/progenitor cells for the AL or IL. Here, we successfully isolated pure CD9/CD81/S100ß/SOX2-positive cells from the IL-side primary niche. They had a higher level of S100ß and SOX2 mRNA and a greater pituisphere forming capacity than those of CD9/CD81/S100ß/SOX2-positive cells isolated from the AL. They also had capacity to differentiate into all types of adenohypophyseal hormone-producing cells, concomitantly with the loss of CD9 expression. Loss of CD9 and CD81 function in CD9/CD81/S100ß/SOX2-positive cells by siRNA treatment impaired prolactin cell differentiation. Consistently, in the pituitary gland of CD9/CD81 double knockout mice, dysgenesis of the MCL and a lower population of prolactin cells were observed. These results suggest that the CD9/CD81/S100ß/SOX2-positive cells in the MCL of the IL-side are potential suppliers of adult core stem cells in the AL.

CX3CL1/CX3CR1-signalling in the CD9/S100ß/SOX2-positive adult pituitary stem/progenitor cells modulates differentiation into endothelial cells.

Approximately 8% of CD9-, S100ß- and SOX2-triple positive (CD9/S100ß/SOX2-positive) stem/progenitor cells in the anterior lobe of the rat pituitary gland have previously been shown to differentiate into endothelial cells in vitro, suggesting that they play a role in vascularisation as tissue-resident vascular precursor cells. In the present study, we focused on chemokine ligands to further characterise the CD9/S100ß/SOX2-positive cells and found that they distinctively express CX3C chemokine ligand 1 (Cx3cl1). Immunohistochemical analysis of the anterior lobe showed that CX3CL1-positive cells comprised 7.8% in CD9-positive cells. By cultivation of the CD9-positive cells on laminin-coated plates, we observed that the expression levels of Cx3cl1 decreased, while those of Sox18, an endothelial cell-progenitor marker, and Cx3cr1, a CX3CL1 receptor, increased. Furthermore, in a rat model of prolactinoma, the most common pituitary tumour, which is accompanied by frequent neo-vasculogenesis in the anterior lobe, we have confirmed a decrease in Cx3cl1 expression and an increase in Cx3cr1 expression, as well as a prominent increase in Sox18 expression. These findings suggest that CX3CL1/CX3CR1 signalling in CD9/S100ß/SOX2-positive cells plays an important role in resupplying endothelial cells for vascular remodelling in the anterior lobe.

Isolation and characterization of cluster of differentiation 9-positive ependymal cells as potential adult neural stem/progenitor cells in the third ventricle of adult rats.

Ependymal cells located above the ventricular zone of the lateral, third, and fourth ventricles and the spinal cord are thought to form part of the adult neurogenic niche. Many studies have focused on ependymal cells as potential adult neural stem/progenitor cells. To investigate the functions of ependymal cells, a simple method to isolate subtypes is needed. Accordingly, in this study, we evaluated the expression of cluster of differentiation (CD) 9 in ependymal cells by in situ hybridization and immunohistochemistry. Our results showed that CD9-positive ependymal cells were also immunopositive for SRY-box 2, a stem/progenitor cell marker. We then isolated CD9-positive ependymal cells from the third ventricle using the pluriBead-cascade cell isolation system based on antibody-mediated binding of cells to beads of different sizes and their isolation with sieves of different mesh sizes. As a result, we succeeded in isolating CD9-positive populations with 86% purity of ependymal cells from the third ventricle. We next assayed whether isolated CD9-positive ependymal cells had neurospherogenic potential. Neurospheres were generated from CD9-positive ependymal cells of adult rats and were immunopositve for neuron, astrocyte, and oligodendrocyte markers after cultivation. Thus, based on these findings, we suggest that the isolated CD9-positive ependymal cells from the third ventricle included tanycytes, which are special ependymal cells in the ventricular zone of the third ventricle that form part of the adult neurogenic and gliogenic niche. These current findings improve our understanding of tanycytes in the adult third ventricle in vitro.

Expression and functions of cluster of differentiation 9 and 81 in rat mammary epithelial cells.

Cluster of differentiation (CD) 9 and CD81 are closely-related members of the tetraspanin family that consist of four-transmembrane domain proteins. Cd9 and Cd81 are highly expressed in breast cancer cells; however, their expression in healthy mammary glands is unclear. In this study, we performed quantitative real-time PCR to analyze the expression levels of Cd9 and Cd81. Histological techniques were employed to identify Cd9- and Cd81-expressing cells in rat mammary glands during pregnancy and lactation. It was observed that Cd9 and Cd81 were expressed in the mammary glands, and their expression levels correlated with mammary gland development. To identify cells expressing Cd9 and Cd81 in the mammary glands, we performed double immunohistochemical staining for CD9 and CD81, prolactin receptor long form, estrogen receptor alpha, or Ki67. The results showed that CD9 and CD81 were co-expressed in proliferating mammary epithelial cells. Next, we attempted to isolate CD9-positive epithelial cells from the mammary gland using pluriBead cell-separation technology based on antibody-mediated binding of cells to beads of different sizes, followed by isolation using sieves with different mesh sizes. We successfully isolated CD9-positive epithelial cells with 96.8% purity. In addition, we observed that small-interfering RNAs against Cd9 and Cd81 inhibited estrogen-induced proliferation of CD9-positive mammary epithelial cells. Our current findings may provide novel insights into the proliferation of mammary epithelial cells during pregnancy and lactation as well as in pathological processes associated with breast cancer.

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-ß2 (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.

Expression of Slug in S100ß-protein-positive cells of postnatal developing rat anterior pituitary gland.

Among heterogeneous S100ß-protein-positive (S100ß-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. S100ß-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 S100ß-GFP transgenic rats expressing specifically green fluorescent protein (GFP) under the control of the S100ß-promoter has allowed us to observe living S100ß-positive cells. In the present study, we first confirmed that living S100ß-positive cells in tissue cultures of S100ß-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 S100ß-positive cells. Interestingly, we detected Slug expression in S100ß-positive cells and its increase together with development in the anterior pituitary. To analyze downstream of SLUG in S100ß-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 S100ß-positive cells express Slug and that its expression is important for subsequent migration and proliferation.

CXCL10/CXCR3 signaling mediates inhibitory action by interferon-gamma on CRF-stimulated adrenocorticotropic hormone (ACTH) release.

Secretion of hormones by the anterior pituitary gland can be stimulated or inhibited by paracrine factors that are produced during inflammatory reactions. The inflammation cytokine interferon-gamma (IFN-γ) is known to inhibit corticotropin-releasing factor (CRF)-stimulated adrenocorticotropin (ACTH) release but its signaling mechanism is not yet known. Using rat anterior pituitary, we previously demonstrated that the CXC chemokine ligand 10 (CXCL10), known as interferon-γ (IFN-γ) inducible protein 10 kDa, is expressed in dendritic cell-like S100ß protein-positive (DC-like S100ß-positive) cells and that its receptor CXCR3 is expressed in ACTH-producing cells. DC-like S100ß-positive cells are a subpopulation of folliculo-stellate cells in the anterior pituitary. In the present study, we examine whether CXCL10/CXCR3 signaling between DC-like S100ß-positive cells and ACTH-producing cells mediates inhibition of CRF-activated ACTH-release by IFN-γ, using a CXCR3 antagonist in the primary pituitary cell culture. We found that IFN-γ up-regulated Cxcl10 expression via JAK/STAT signaling and proopiomelanocortin (Pomc) expression, while we reconfirmed that IFN-γ inhibits CRF-stimulated ACTH-release. Next, we used a CXCR3 agonist in primary culture to analyze whether CXCL10 induces Pomc-expression and ACTH-release using a CXCR3 agonist in the primary culture. The CXCR3 agonist significantly stimulated Pomc-expression and inhibited CRF-induced ACTH-release, while ACTH-release in the absence of CRF did not change. Thus, the present study leads us to an assumption that CXCL10/CXCR3 signaling mediates inhibition of the CRF-stimulated ACTH-release by IFN-γ. Our findings bring us to an assumption that CXCL10 from DC-like S100ß-positive cells acts as a local modulator of ACTH-release during inflammation.

Proton receptor GPR68 expression in dendritic-cell-like S100ß-positive cells of rat anterior pituitary gland: GPR68 induces interleukin-6 gene expression in extracellular acidification.

S100ß-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 S100ß-positive cells are composed of populations such as stem/progenitor cells, epithelial cells, astrocytes and dendritic cells. Recently, we succeeded in separating S100ß-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 ß-6) in the round type. Here, we further investigate the function of the subpopulation of S100ß-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 S100ß-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, S100ß-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 S100ß-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 S100ß-protein-positive cells (S100ß-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 S100ß-positive cells and those expressing Cxcr3 amongst adrenocorticotropic hormone (ACTH)-producing cells. We also investigated Cxcl10 expression in subpopulations of S100ß-positive cells. We separated cultured S100ß-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 S100ß-positive cells. Thus, CXCL10 produced by a subpopulation of S100ß-positive cells probably exerts an autocrine/paracrine effect on S100ß-positive cells and ACTH-producing cells in the anterior lobe.

Estrogen suppresses retinaldehyde dehydrogenase 1 expression in the anterior pituitary glands of female rats.

Retinoic acid (RA) plays a critical role in cell growth and tissue development. RA is also a regulating factor of pituitary function. RA is synthesized from retinoids through oxidation processes. The oxidation of retinal to RA is catalyzed by the retinaldehyde dehydrogenases (RALDHs), including RALDH1, RALDH2 and RALDH3. Recently, we demonstrated that RALDH1 is expressed in the anterior pituitary glands of adult male rats. However, the expression of RALDH1 in the female pituitary gland and the regulation of RALDH1 expression have not been determined. Therefore, we examined the expression of RALDH1 mRNA in the pituitary glands of adult female rats. By in situ hybridization with digoxigenin-labeled cRNA probes and quantitative real-time PCR analysis, we found that the expression level of RALDH1 was significantly lower in estrus as compared to proestrus, metestrus and diestrus. RALDH1 mRNA levels increased after ovariectomy and decreased remarkably after a 1-week treatment with 17beta-estradiol implants. Estradiol (0.01-100 microg per rat) dose-dependently decreased the expression of RALDH1 in the pituitary glands after 24 hours of subcutaneous administration. These results clearly show that RALDH1 mRNA expression is suppressed by estrogen. We speculate that the generation of RA is regulated by estrogen and that RA plays a role in the estrus cycle through paracrine and/or autocrine mechanisms in the anterior pituitary gland of female rats.

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.

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

S100ß protein and SOX2-double positive (S100ß/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 S100ß/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 S100ß/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.

Distinctive localization of N- and E-cadherins in rat anterior pituitary gland.

In the rat anterior pituitary gland, folliculo-stellate cells aggregate preferably to form pseudofollicles, and each type of hormone-producing cell shows adhesive affinity with particular types of heterologous hormone-producing cells. Distribution of cadherin types in the rat anterior pituitary was examined immunohistochemically to clarify the unique cell arrangements caused by homologous and heterologous affinities among cells. N- and E-cadherins were detected continuously along cell membranes, while P-cadherin was not. N- and E-cadherins showed distinct isolation in localization, with N-cadherins localized in hormone-producing cells of distal and intermediate lobes in various amounts, and E-cadherins limited to folliculo-stellate cells and marginal layer cells facing the residual lumen of Rathke's pouch. A similar distribution of cadherins was observed in cell clusters of primary cultured anterior pituitary cells. These findings suggest that differential expression of cell adhesion molecules may be partially responsible for localization of hormone-producing cells and folliculo-stellate cells.

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-microm-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.

Fetal development of vomeronasal system in the goat.

Our previous study morphologically revealed that the adult goat vomeronasal (VN) system was different from the rodent and opossum one, and at least two types of VN systems exist in mammals. However, it remains unknown whether the developments in both types of VN systems are ontogenetically distinct and when the goat VN system is established. In this study, we morphologically observed the fetal development of the goat accessory olfactory bulb (AOB) and VN neuron. In the fetus, Gi2-expressing VN terminals terminated at glomeruli throughout the AOB, and no immunoreactivities for Go were detected in the nerve terminals reaching into AOB. The layer structure of AOB rapidly developed in the latter half of gestation. In the VN organ (VNO), at the middle stage of gestation, the dendritic processes of VN neuron were exposed in the VN lumen, and scattered and thin microvilli existed on the protrusion of the VN neuron. In the apical part of dendritic processes, no clear vesicle existed. However, the immunohistochemistry of an olfactory marker protein (OMP) revealed that a few VN neurons with OMP exist in VN sensory epithelium (VSE) before birth, although marked immunoreactivities were detected in adult VSE. Fetal VN neurons appeared to be underdeveloped. These results suggest that the goat VN system is ontogenetically distinct from the rodent and opossum VN systems, and is underdeveloped before birth. The goat VN system will develop and mature during the early postnatal period similar to the rodent and opossum VN systems.