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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.

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 S100β 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 S100β/SOX2-positive cells of primary and secondary niches (named CD9/CD81/S100β/SOX2-positive cell), and the cells located in the AL-side niches exhibit plasticity and multipotency. However, it is unclear whether CD9/CD81/S100β/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/S100β/SOX2-positive cells from the IL-side primary niche. They had a higher level of S100β and SOX2 mRNA and a greater pituisphere forming capacity than those of CD9/CD81/S100β/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/S100β/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/S100β/SOX2-positive cells in the MCL of the IL-side are potential suppliers of adult core stem cells in the AL.

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.

Approximately 8% of CD9-, S100β- and SOX2-triple positive (CD9/S100β/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/S100β/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/S100β/SOX2-positive cells plays an important role in resupplying endothelial cells for vascular remodelling in the anterior lobe.

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.

The adenohypophysis is composed of the anterior and intermediate lobes (AL and IL), and secretes important hormones for growth, sexual development, metabolism, and reproduction. In the marginal cell layer (MCL) facing Rathke's cleft between the IL and AL, cluster of differentiation (CD) 9-, CD81-, S100β-, and SOX2-quadruple positive (CD9/CD81/S100β/SOX2-positive) cells in the adult IL are settled as tissue-resident stem/progenitor cells supplying hormone-producing cells to the AL. However, it is unclear how CD9/CD81/S100β/SOX2-positive cells in the IL-side MCL migrate into the AL across Rathke's cleft. In the present study, we performed chimeric pituitary tissue culture using S100β/GFP-transgenic rats and Wistar rats, and traced the footprint of S100β/GFP-expressing cells. We detected IL-side S100β/GFP-expressing cells in the AL tissue, demonstrating that these cells migrate from the IL to the AL. However, the cells failed to migrate in the opposite direction. Consistently, scanning electron microscopic analysis revealed well-developed cytoplasmic protrusions in the IL-side MCL, but not in the AL-side MCL, suggesting that IL-side CD9/CD81/S100β/SOX2-positive cells had higher migratory activity. We also searched for a specific marker for IL-side CD9/CD81/S100β/SOX2-positive cells and identified tetraspanin 1 (TSPAN1) from microarray analysis. Downregulation of Tspan1 by specific siRNA impaired cell migration and significantly reduced expression of snail family transcriptional repressor 2 (Slug), a marker of epithelial-mesenchymal transition (EMT). Therefore, CD9/CD81/S100β/SOX2-positive cells in the IL-side MCL can be stem/progenitor cells that provide stem/progenitor cells to the AL-side MCL via SLUG-mediated EMT and cell migration.

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.

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 S100β protein-positive (DC-like S100β-positive) cells and that its receptor CXCR3 is expressed in ACTH-producing cells. DC-like S100β-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 S100β-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 S100β-positive cells acts as a local modulator of ACTH-release during inflammation.

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.

To determine the morphological basis for the neurotrophic effects of brain-derived neurotrophic factor (BDNF) in the primary olfactory pathway (POP), tyrosine kinase receptor B (TrkB), a membrane-bound receptor for BDNF, was identified and localized in axons of olfactory receptor cells (ORC) of neonatal rat olfactory mucosa using immuno-histochemical and -cytochemical techniques. Initially, the immunospecificity of an anti-TrkB antibody that had been used as a specific antibody for full-length TrkB was confirmed in the olfactory mucosa. Then, a combination of a reduced osmium-LR-White and post-embedding immunogold technique was applied to ORC axons in the lamina propria just beneath the olfactory epithelium. Immunogold particles, which indicate TrkB immunoreactivity, were noted either in close association with the plasma membranes of ORC axons, and designated plasma-lemmal (PL), or within their cytoplasm, and designated cytoplasmic (CP). Most PL particles were seen in the CP portion of the axonal plasma membranes, suggesting that the anti-TrkB antibody binds to the membrane-inserted TrkB that acts as a functional receptor. Some CP particles were on vesicular structures. Quantitative analysis demonstrated that the ratio of CP to PL particles was 7:3, and this ratio was constant between animals examined (n = 5). Because membrane proteins are wrapped in vesicles and transported within the axonal cytoplasm and inserted into the plasma membrane to function there, the present study suggests that TrkB is transported within the cytoplasm of ORC axons and is positioned as a functional receptor for BDNF in their membranes.

Binding sites of Griffonia simplicifolia I-B4 isolectin (GS-I-B4), which recognizes terminal alpha-galactose residues of glycoconjugates, were examined in the juxtaluminal region of the rat vomeronasal sensory epithelium and its associated glands of the vomeronasal organ, using a lectin cytochemical technique. Lowicryl K4M-embedded ultra-thin sections, which were treated successively with biotinylated GS-I-B4 and streptavidin-conjugated 10 nm colloidal gold particles, were observed under a transmission electron microscope. Colloidal gold particles, which reflect the presence of terminal alpha-galactose-containing glycoconjugates, were present in vomeronasal receptor neurons in the sensory epithelium and secretory granules of acinar cells of associated glands of the epithelium. Quantitative analysis demonstrated that the density of colloidal gold particles associated with sensory cell microvilli that projected from dendritic endings of vomeronasal neurons was considerably higher than that of microvilli that projected from neighboring sustentacular cells. The same was true for the apical cytoplasms of these cells just below the microvilli. These results suggest that of the sensory microvilli and dendritic endings contained a much larger amount of the alpha-galactose-containing glycoconjugates, compared with those in sustentacular microvilli. Further, biochemical analyses demonstrated several vomeronasal organ-specific glycoproteins with terminal alpha-galactose.

Brain-derived neurotrophic factor (BDNF) is one of the neurotrophins, and known to facilitate differentiation, growth and maturation of neurons. BDNF binds to the high-affinity receptor, tyrosine kinase receptor B (TrkB) to initiate signal transduction (Korsching, 1993; Lindsay et al., 1994). Olfactory and vomeronasal receptor neurons (ORNs and VRNs, respectively) are chemoreceptors located in the nasal cavity of most mammalian species (Graziadei, 1977). In the olfactory epithelium (OE) lining the olfactory mucosa (OM), progenitor cells differentiate into immature and mature ORNs; a single dendrite extending from the apical pole of each soma reaches the surface of the OE to form a dendritic ending, and a single axon from the base of the soma runs downward to pass through the basement membrane of the OE to reach the brain. Similar bipolar neurons are also contained in the vomeronasal sensory epithelium (VSE) of the vomeronasal organ (VNO), and designated as VRNs or vomeronasal sensory cells/ neurons (Graziadei, 1977; Takami, 2002). Although several neurotrophic factors including neurotrophins are thought to be involved in the development, maturation, and regeneration of ORNs (Carter and Roskams, 2002; Schwob, 2002), the functional roles of BDNF remain to be understood. In the case of the VRNs, we were the first to report the distribution of BDNF and TrkB at both domestic (Takami et al., 2001) and international (Takami and Nishiyama, 1997b) meetings. In this paper, we present summary of our recent studies concerning BDNF and TrkB in the rat OM and VNO. Our overall research goal is to clarify functional roles of BDNF in the differentiation and maturation of ORNs and VRNs.