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Keywords:

  • cadherin;
  • rat;
  • anterior pituitary gland;
  • folliculo-stellate cell;
  • immunohistochemistry

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

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. Anat Rec Part A, 288A:1183–1189, 2006. © 2006 Wiley-Liss, Inc.

The rat anterior pituitary gland consists of five types of hormone-producing cells, together with folliculo-stellate cells that do not produce hormones. These cells form a unique topography caused by homologous and heterologous affinities between cell types (Nakane, 1970; Horvath et al., 1977; Noda et al., 2001). Folliculo-stellate cells aggregate homologeously to form pseudofollicles. Conversely, each type of hormone-producing cell shows affinities with specific heterologous hormone-producing cells. Moreover, when completely dispersed cells of the anterior pituitary are applied to static culture, cells gradually gather to form clusters. Observation of cell clusters under electron microscopy has indicated homologous reassembly of folliculo-stellate cells and the presence of cell junctions among folliculo-stellate cells, but not among hormone-producing cells, as seen in tissue observation (Noda et al., 2003). These results indicate the involvement of various cell adhesion molecules in tissue construction of the anterior pituitary. However, despite detailed morphological studies, cell adhesion molecules generally have not been studied in the anterior pituitary gland.

Cadherins are a family of cell surface glycoproteins that mediate calcium-dependent cell-to-cell adhesion. Numerous types of cadherins have been identified and shown to regulate morphogenesis through homophilic binding (Nose et al., 1988; Takeichi, 1990). In adults, these molecules are essential for stable cell attachments to maintain the systematic tissue structure. Recently, large amounts of data have indicated that cadherins can affect cell form, growth, motility, and functions, not only due to mechanical attachment of cells, but also by activating intracellular signaling pathways (Pece and Gutkind, 2000). Expression of cadherins in the anterior pituitary gland has been confirmed (Sugimoto et al., 1996; Heinrich et al., 1999; Rubinek et al., 2003), but insufficient morphological techniques have been applied to this investigation.

The present study was the first to investigate immunohistochemically the precise distribution of cadherins and to clarify relationships between these cell adhesion molecules and local histology of the rat anterior pituitary using tissues and primary cultured cells.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

Animals

Male Sprague-Dawley rats (Japan SLC, Shizuoka, Japan) were used at 8 to 10 weeks old and weighed 250–300 g. They were supplied food and water ad libitum and kept under light cycle of 12-hr light and 12-hr darkness. All animals were treated in accordance with the Guidelines for Animal Experimentation of Jichi Medical University based on NIH Guidelines for the Care and Use of Laboratory Animals.

Immunostaining of Tissue

Cryosection.

Under deep Nembutal anesthesia, rats were perfused with 4% paraformaldehyde and 0.1% glutaraldehyde in 25 mM phosphate buffer (pH 7.4, 4°C) for 5 min through the left ventricle, then immersed in the same fixative for a total of 60 min. Using 20% polyvinylpyrrolidone (Sigma Chemical, MO) and 1.84 M sucrose in phosphate-buffered saline (PBS; pH 7.2) as antifreeze agents, pieces of anterior pituitary gland were frozen in liquid nitrogen. Frozen sections (1.5 or 2 μm thick) were obtained using a Reichert-Nissei Ultracut S ultramicrotome (Leica Aktiengesellschaft, Vienna, Austria). Sections were immersed in 3% H2O2 for 3 min at room temperature prior to immunohistochemistry.

Paraffin-embedded section.

Excised pituitary gland was fixed overnight at 4°C in Bouin's fluid or for 6 hr at 4°C in sublimated formalin. Fixed tissues were processed routinely and embedded in Pathoprep embedding media (Wako Pure Chemical Industries, Osaka, Japan). Frontal sections of 2 μm thickness were prepared.

Immunohistochemistry.

Sections were incubated in 0.05% citraconic anhydride solution (Nissin EM, Tokyo, Japan) for 60 min at 95°C for antigenic retrieval. After immersion in PBS containing 2% normal goat serum for 20 min at room temperature, sections were incubated in PBS with primary antibodies overnight at 30°C. After washing with PBS, sections were incubated in PBS with biotinylated secondary antibodies for 30 min at 30°C. The primary and secondary antibodies used are shown in Tables 1 and 2, respectively. Absence of observable nonspecific reaction was confirmed using normal animal serum. The ABC method (Vector Laboratories, CA) using 3,3′-diaminobenzidine (DAB; Dojindo Laboratories, Kumamoto, Japan) as substrate was utilized. For double immunostaining, a pair out of FITC, Alexa Fluor 488 and Texas Red-labeled secondary antibodies was utilized. After all processes of immunostaining against the first antigen were completed using monoclonal primary antibody, another immunostaining against second antigen was performed using polyclonal primary antibody. Sections were observed through an AX80TR fluorescent microscope (Olympus, Tokyo, Japan) and imaged using a DP70 system (Olympus) with aid of Photoshop software (Adobe Systems, CA).

Table 1. Primary antibodies
AntigenImmunized animalDilution factorSupplier
in vivoin vitro
  1. GH, growth hormone; LH, luteinizing hormone; ACTH, adrenocorticotrophic hormone; TSH, thyroid stimulating hormone.

human N-cadherinrabbit1:40-1601:100IBL, Gunma, Japan
human E-cadherinrabbit1:25-1601:100Santa Cruz Biotechnology, CA, USA
human E-cadherinmouse1:2001:200BD Biosciences, CA, USA
human P-cadherinrabbit1:1601:100Santa Cruz Biotechnology, CA, USA
rat GHrabbit1:25,600 Dr. K. Wakabayashi, Gunma Univ., Japan
ovine LH β-subunitrabbit1:25,600 Dr. K. Wakabayashi, Gunma Univ., Japan
rat prolactinrabbit1:25,600 NIH, MD, USA
porcine ACTH 1-39rabbit1:12,800 Dr. H. Nakamura, Hokkaido Univ., Japan
rat TSH β-subunitrabbit1:25,600 NIH, MD, USA
S-100 β-subunitmouse1:80 JIMRO, Gunma, Japan
Table 2. Secondary antibodies
AntigenImmunized animalLabelSupplier
rabbit IgGgoatbiotinVector Laboratories, CA, USA
rabbit IgGgoatFITCICN Pharmaceuticals, CA, USA
rabbit IgGgoatTexas RedICN Pharmaceuticals, CA, USA
mouse IgGgoatAlexa Fluor 488Molecular Probes, OR, USA

Immunostaining of Cultured Cells

Primary culture.

Rats were perfused with Ca2+- and Mg2+-free (CMF) Hanks' solution under deep Nembutal anesthesia. Anterior pituitary glands were excised and minced into pieces that were incubated in CMF Hanks' solution containing 1% trypsin (Life Technologies, MD) and 0.2% collagenase (Nitta Gelatin, Osaka, Japan) for 15 min at 37°C. Thereafter, tissue pieces were incubated in the same solution containing 5 μg/ml of DNase I (Boehringer-Mannheim, Mannheim, Germany) for 5 min at 37°C. The digest was incubated in Hanks' solution containing 5 mM ethylenediamine tetraacetic acid (Wako Pure Chemical Industries) for 5 min at 37°C. After washing, cells were dispersed in CMF Hanks' solution by gentle pipetting, then dispersed cells were separated from debris by filtering through nylon mesh (Becton Dickinson Labware, NJ). After centrifugation, cells were resuspended in Medium 199 (Life Technologies) supplemented with 10% fetal bovine serum (Sigma Chemical), 100 u/ml penicillin, and 100 μg/ml streptomycin, plated on eight-well glass chamber slides (0.8 cm2/well; Nalge Nunc International, NY) at a density of 1 × 105 cells/400 μl/cm2, and cultured for 3 days at 37°C in a humidified atmosphere of 5% CO2 and 95% air.

Immunocytochemistry.

Cultured cells were rinsed with Hanks' solution and fixed with 4% paraformaldehyde in 25 mM phosphate buffer (pH 7.4) for 30 min at room temperature. Fixed cells were microwave-irradiated in 0.05% citraconic anhydride solution for 5 min for antigenic retrieval. The procedure for immunostaining was essentially the same as in immunohistochemistry of the tissue. Specimens were observed using a CSU10 confocal laser microscope (Yokogawa Electric, Tokyo, Japan).

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

Detection of Cadherin Types in Anterior Pituitary Gland In Vivo and In Vitro

Localization of N-, E-, and P-cadherin within the anterior pituitary was examined immunohistochemically using cryosections. Positive signals were detected for N- and E-cadherin, but not for P-cadherin (Fig. 1). The major positive signals of N- and E-cadherins appeared continuously along the cell membrane. N-cadherin was seen on the vast majority of anterior pituitary cells with various signal strengths, while E-cadherin was limited to scattered cell clusters. In addition, expressions of N-, E-, and P-cadherin in reassembled anterior pituitary cell clusters in primary culture were examined immunohistochemically under confocal laser microscopy. Positive signals were observed for N- and E-cadherin in the vicinity of cell membrane, except for the free surface of the cluster (Fig. 2). Distribution patterns of N- and E-cadherin were comparable with those in tissue (Fig. 1).

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Figure 1. Immunohistochemical staining of N- (a), E- (b), and P- (c) cadherin on cryosections of rat anterior pituitary. An ABC method using DAB as substrate was utilized. Sections were counterstained with hematoxylin. Scale bar = 10 μm.

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Figure 2. Immunohistochemical staining of N- (a), E- (b), and P- (c) cadherin on primary cultured anterior pituitary cells. FITC-conjugated secondary antibody was utilized. Images were captured by confocal laser microscopy. Scale bar = 10 μm.

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Distribution of N- and E-Cadherin in Pituitary Gland

Based on the above results, distribution of N- and E-cadherin was examined using paraffin-embedded sections over a wide area, including distal and intermediate lobes. Positive signals for N-cadherin appeared on the majority of cells in distal and intermediate lobes (Fig. 3a). Marginal layer cells that faced the residual lumen of Rathke's pouch were the exception, with few cells in the layer exhibiting N-cadherin expression. In contrast with the distribution of N-cadherin, positive signals for E-cadherin appeared on limited cells of the distal lobe and prominently on marginal layer cells, particularly on the side of the intermediate lobe (Fig. 3b). Allopatry of N- and E-cadherin was clearly shown on double immunostaining (Fig. 4a). Some exceptional colocalization of N- and E-cadherin may exist in the distal lobe, particularly near the residual lumen of Rathke's pouch and the so-called posterolateral wing. In addition, differential cadherin expression of cells was shown in double immunostaining of primary cultured cells (Fig. 4b). Cells showed adhesion following type of cadherin and E-cadherin-expressing cells aggregated with one another.

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Figure 3. Immunohistochemical staining of N- (a) and E- (b) cadherin on paraffin-embedded sections. An ABC method using DAB as substrate was utilized. Sections were counterstained with hematoxylin. PI, pars intermedia; PD, pars distalis. Scale bar = 50 μm.

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Figure 4. Double immunostaining of N-cadherin (Texas Red) and E-cadherin (Alexa Fluor 488) in vivo (a) and in vitro (b). PI, pars intermedia; PD, pars distalis. Scale bars = 100 μm (a); 10 μm (b).

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Expression of N-Cadherin on Types of Hormone-Producing Cells

Levels of N-cadherin expression on five types of hormone-producing cells were examined immunohistochemically in cryosections. Pairs of mirror sections were prepared and stained immunohistochemically for N-cadherin and types of hormone. Results of N-cadherin immunostaining are shown in Figure 5 with markers of cell types identified using countersections. N-cadherin was expressed in all types of hormone-producing cells with varying signal strength. Growth hormone (GH) cells were strongly stained, particularly where GH cells adhered to each other. Other types of hormone-producing cells tended to stain weakly compared with GH cells.

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Figure 5. Immunohistochemical staining of N-cadherin on types of hormone-producing cells in rat anterior pituitary. An ABC method using DAB as substrate was utilized. Cell type as identified by counterimmunostaining of the mirror section is indicated by asterisks. GH, growth hormone cell; LH, luteinizing hormone cell; TSH, thyroid-stimulating hormone cell; PRL, prolactin cell; ACTH, adrenocorticotrophic hormone cell. Scale bar = 10 μm.

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Expression of Cadherin on Folliculo-Stellate Cells

Concerning the distinctive distribution pattern of E-cadherins shown above, expressions of N- and E-cadherin on folliculo-stellate cells were examined by double-immunostaining using paraffin-embedded sections. Folliculo-stellate cells were detected by marker protein S-100. N-cadherin was not distributed among folliculo-stellate cells (Fig. 6a). Conversely, distribution of E-cadherin was largely limited to folliculo-stellate cells (Fig. 6b). Distribution of cadherin on the agglutination surface of folliculo-stellate and hormone-producing cells was obscure.

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Figure 6. Double immunohistochemical staining of N- (a) and E- (b) cadherin against S-100 marker protein for folliculo-stellate cells on paraffin-embedded sections. Cadherins were stained by Texas Red and S-100 protein by Alexa Fluor 488. Scale bar = 10 μm.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

Cadherins are a family of cell surface glycoproteins that mediate calcium-dependent cell-to-cell adhesion in solid tissues. Various isoforms are distributed in a tissue-specific manner. Highly specific homophilic interactions between extracellular domains of identical cadherins on neighboring cells play a key role in cell sorting and histogenesis (Takeichi, 1990; Ivanov et al., 2001; Wheelock and Johnson, 2003), with cells expressing the same cadherins tending to aggregate preferentially, and those expressing different cadherins tending to segregate. These cell adhesion molecules are also involved in the formation of zonula adherens (Yap et al., 1997).

The present study focused on types of cadherin in the anterior pituitary gland, and results can be summarized as follows. In the adult rat adenohypophysis, N- and E-cadherin represent the major cadherin types, while P-cadherin may be absent. N- and E-cadherins show distinct isolation in localization, with N-cadherin expressed in hormone-producing cells of the distal and intermediate lobes and E-cadherin expressed in folliculo-stellate cells and marginal layer cells. Among the various cadherins, N- and E-cadherin belong to a group of classic type I cadherins. In general, E-cadherin, named after epithelial cadherin, distributes widely on epithelial tissues, while N-cadherin, named after neuronal cadherin, distributes in a limited manner to the nervous system, cardiac muscle, crystalline, etc., in adult animals (Hatta et al., 1987). Location of N-cadherin in the adenohypophysis seems unique and may be associated with early development during the induction of Rathke's pouch from the primordial area just anterior to the neural plate (Kouki et al., 2001).

The finding that marginal layer cells and folliculo-stellate cells commonly expressed E-cadherin but not N-cadherin may suggest an intimate relationship between these cell types. Yoshimura et al. (1977) described this relationship in electron microscopic observations, noting cytological properties of immaturity and cilia on the free surfaces of both cells. In contrast, all types of hormone-producing cells appeared to express N-cadherin, although amounts of expression varied greatly from cell to cell and cell type to cell type. GH cells expressed N-cadherin most strongly, and this may be associated with the fact that GH cells tend to aggregate homogeneously compared with other hormone-producing cells (Noda et al., 2001). Connecting the differing amounts of N-cadherin expression with heterophilic cell affinities shown by hormone-producing cells in the anterior pituitary may yield interesting results. Noda et al. (2001) statistically showed cell affinities between GH and adrenocorticotrophic hormone (ACTH) cells and between luteinizing hormone (LH) and prolactin (PRL) cells. However, our triple-immunostaining observation of N-cadherin and pairs of hormone-producing cells found no relation between expression of N-cadherin and preferential heterophilic attachments of hormone-producing cells (data not shown). Recently, various data have indicated that cadherins can affect cell form, motility, and proliferation by activating intracellular signaling (Pece and Gutkind, 2000). Rubinek et al. (2003) reported that in the pituitary gland, binding of cell surface N-cadherin with external N-cadherin may affect GH expression in somatotropes. Cadherin is considered to be involved in not only mechanical cell attachments, but also information transfer between neighboring cells and functional modification of cells. Moreover, cadherin-mediated adhesion is known to be regulated by various extra- and intracellular signals (Bradley et al., 1993; Bracke et al., 1994; Brabant et al., 1995). The differing levels of N-cadherin expression seen in the present study may not reflect heterologous cell preferences, but such kinds of cell activities.

The allopatry of N- and E-cadherin shown in the present study suggests the distribution of hormone-producing cells and folliculo-stellate cells in the anterior pituitary. Types of cadherin mediate cell sorting through homophilic interactions. Nose et al. (1988) showed that when heterogeneous cell populations with different expressions of cadherins were cocultured, aggregation was seen for cells expressing identical cadherins on the surfaces. A similar role of cadherins may enable folliculo-stellate cells to separate from hormone-producing cells and aggregate homologously to construct pseudofollicles. In addition, zonula adherens is known to be specific to folliculo-stellate cells in the pituitary gland (Soji et al., 1997). Zonula adherens is a specialized form of cadherin-mediated cell-to-cell adhesion, but cadherins can also mediate adhesion without the formation of morphologically apparent adhesive junctions (Yap et al., 1997). E-cadherin, but not N-cadherin, is apparently associated with the formation of zonula adherens in the anterior pituitary. However, careful examination of these concepts is warranted, as the existence of cell adhesion molecules other than N- and E-cadherin, such as neural cell adhesion molecule (NCAM) (Berardi et al., 1995) and PB-cadherin (Sugimoto et al., 1996), have been reported in the anterior pituitary.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED