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Keywords:

  • anterior pituitary gland;
  • topographic cell affinity;
  • primary monolayer culture;
  • immunohistochemistry;
  • confocal laser microscopy

Abstract

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

Hormone-producing cells in the rat anterior pituitary gland are not randomly distributed; rather, there are specific topographic affinities among five cell types (Noda et al., Acta Histochem. Cytochem. 2001;34:313–319). In this study we reconstructed these affinities, at least partially, in primary monolayer culture. Pituitary cells collected from adult male rats were enzymatically dispersed and cultured for 72 hr at a density of 1 × 105 cells/cm2. We double-immunostained cells using antibodies against hormones, and then used confocal laser microscopy to examine the ability of the cells to attach to each other. We also statistically analyzed the affinity of all combinations of the five types of hormone-producing cells. We observed clusters by electron microscopy to identify junctional complexes between the cells. Confocal laser microscopy indicated that the features and attachment patterns of hormone-producing cells in vivo were similar to those in vitro. Statistical analyses revealed that the rates at which the five types of hormone-producing cells attached to growth hormone (GH)-, prolactin (PRL), and luteinizing hormone (LH)-producing cells were unequal, which suggests there are specific topographic affinities. The specific rates of adrenocorticotropic hormone (ACTH)-producing cell attachment to GH cells, LH to PRL cells, and PRL to LH cells were high, whereas that of PRL attachment to PRL cells was low. In addition, the rates correlated with the data from our previous in vivo study. Ultrastructural observations revealed few junctional complexes between hormone-producing cells. These results indicate that anterior pituitary hormone-producing cells can attach to specific types of cells by means of specific and/or nonspecific adhesion factors, and can reconstruct the topographic nature of the pituitary gland. Anat Rec Part A 272A:548–555, 2003. © 2003 Wiley-Liss, Inc.

The anterior pituitary is a multifunctional gland that comprises a mixture of hormone-producing cells. Immunohistochemical studies of the distribution of such cells in the pituitary of various animals have shown that the localization of each cell is species-specific (Doerr-Schott, 1980). In rodents, five types of hormone-producing cells appear in a temporally and spatially specific fashion (Watanabe and Daikoku, 1979) via signals from the ventral diencephalon and oral ectoderm (Scully and Rosenfeld, 2002), but localize almost at random within the entire adult pituitary gland (Nakane, 1970; Baker and Gross, 1978). These cells, like folliculo-stellate (F-S) cells, are distributed with specific topographic affinity in the rat anterior pituitary. Nakane (1970) found high topographic affinity between growth hormone (GH)- and adrenocorticotropic hormone (ACTH)-producing cells. Siperstein and Miller (1970) and Yoshimura and Nogami (1981) published images of an ACTH cell adjacent to a GH cell. Nakane (1970) and Nogami and Yoshimura (1982) found that individual large, oval luteinizing hormone (LH)-/follicle-stimulating hormone (FSH)-producing cells are frequently surrounded by cup-shaped prolactin (PRL)-producing cells. Gon (1987) observed that F-S cells form clusters, and Soji and Herbert (1989) showed images of clustered F-S cells surrounding the lumen. In addition, PRL cells (Nogami and Yoshimura, 1982; Shirasawa et al., 1985) or thyroid-stimulating hormone (TSH)-producing cells (Yashiro et al., 1981) sometimes form clusters. These morphological features should provide a suitable environment for cell-to-cell communication in the pituitary gland. However, when these features are identified by electron microscope, their interpretation is subjective according to the viewer. The interpretation of these data become standardized by observing a wide area of the gland by light microscopy after double-immunostaining the cells, and by statistical analysis (Noda et al., 2001).

Enzymatically dispersed pituitary cells can form clusters in primary monolayer cultures as well as in vivo. Topographic affinity in clusters merits further study, as few reports have examined this issue. Wilfinger et al. (1984) showed that LH cells locate in close proximity to PRL cells when seeded at high density. Allaerts et al. (1991) demonstrated that PRL and LH cells juxtapose in enriched suspension culture. However, the topographic affinities among all types of hormone-producing cells in vitro have not been reported, so the relationship between in vivo and in vitro affinities has remained obscure. We therefore used confocal microscopy to study the affinities of all types of double-immunostained rat anterior pituitary hormone-producing cells in primary monolayer culture. We also examined cell-to-cell junctions in clusters by electron microscopy.

MATERIALS AND METHODS

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

Cell Culture

Male Sprague-Dawley rats were purchased from Japan SLC, Inc. (Shizuoka, Japan). When the rats were 8–10 weeks old (and weighed 250–300 g), they were perfused with Ca2+- and Mg2+-free (CMF) Hank's solution under Nembutal anesthesia. The anterior pituitary glands were excised and minced into small pieces that were incubated in CMF Hank's solution containing 1% trypsin (Life Technologies, Inc., New York, NY) and 0.2% collagenase (Nitta Gelatin Inc., Osaka, Japan) for 20 min at 37°C. Thereafter, the pieces were incubated in the same solution containing 5 μg/ml of DNase I (Boehringer-Mannheim, Mannheim, Germany) for 5 min at 37°C. After the digest was washed with CMF Hank's solution, it was incubated in the same solution containing 0.3% ethylenediaminetetraacetic acid (Wako Pure Chemicals, Osaka, Japan) for 5 min at 37°C. Dispersed cells were separated from debris by centrifugation, rinsed and resuspended in CMF Hank's solution by pipetting, and then filtered through nylon mesh (Becton Dickinson Labware, Franklin Lakes, NJ). The filtered cells were plated on eight-well glass chamber slides (1 cm2/well; Asahi Techno Glass, Chiba, Japan) at a density of 1 × 105 cells/cm2 in 400 μl of Medium 199 with Earle's salts (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (Sigma, St. Louis, MO). They were then cultured for 72 hr at 37°C in a humidified atmosphere of 5% CO2 and 95% air.

Time-Lapse Observation of the Cluster Formation

The cells were cultured under the conditions described above, in a CO2 gas culture chamber (Sankei Co. Ltd., Tokyo, Japan) with a thermostat (Kokensha Engineering Co. Ltd., Tokyo, Japan), and examined by phase contrast microscopy (Diaphot-TMD; Nikon Corporation, Tokyo, Japan). Cluster formation was monitored using a time-lapse VTR system (Sankei Co. Ltd.) for 72 hr.

Immunofluorescence Staining

Cultured cells fixed with 10% buffered formalin for 10 min at room temperature were first immersed in phosphate-buffered saline (PBS, pH 7.2) containing 1% Triton X-100 (Sigma), and then in normal goat serum for 1 hr at 30°C to reduce nonspecific immunoreactivity. Incubation with primary antibodies for 2 hr at 30°C was followed by a wash with PBS containing 1% Triton X-100, an incubation with secondary antibodies for 1 hr at 30°C, and another wash with PBS containing 1% Triton X-100. The specimens were immunostained with another species of antibody, as described above.

Antibodies

Rabbit polyclonal antibodies against rat GH (diluted 1:1,600), rat PRL (1:100), and ovine LH β subunit (1:3,200) were donated by Dr. K. Wakabayashi (Biosignal Research Center, Gunma University, Gunma, Japan). Rabbit polyclonal antibody against porcine ACTH 1–39 (1:1,600) was donated by Dr. F. Nakamura (Hokkaido University, Sapporo, Japan). Rabbit polyclonal antibody against rat TSH β subunit (1:3,200) and guinea pig antibody against rat LH β subunit (1:1,000) were obtained from the National Institutes of Health (Bethesda, MD). Guinea pig polyclonal antibody against human GH (1:200) was purchased from Biogenesis Ltd. (Poole, UK). Mouse monoclonal antibodies against rat PRL (1:400) and human ACTH C-terminal (1:200) were purchased from Chemicon International Inc. (Temecula, CA) and Cymbus Biotechnology Ltd. (Hampshire, UK), respectively. Nonspecific bindings of each primary antibody were checked using nonimmune rabbit or guinea pig serum and mouse IgG.

Goat polyclonal antibody against rabbit IgG labeled with Texas Red (1:50) was purchased from ICN Pharmaceuticals Inc. (Aurora, OH). An antibody against guinea pig IgG labeled with fluorescein isothiocyanate (FITC, 1:100) and mouse IgG labeled with FITC (1:100) were purchased from EY Laboratories Inc. (San Mateo, CA). The absence of cross reactions in double immunostaining was confirmed in preliminary experiments. All antibodies were diluted with PBS containing 0.1% NaN3.

Observation of Topographic Cell Affinity

The specimens were mounted with Vectashield (Vector Laboratories, Burlingame, CA), and clusters were observed with a confocal laser microscope (Yokogawa Electric Co., Tokyo, Japan) at wavelengths of 488 and 568 nm for FITC and Texas Red, respectively, to confirm the frequency with which the specific cell types attached. The same field was captured at both wavelengths and merged with the use of a computer program (MetaMorph, Universal Imaging Co., Downingtown, PA). The images were further processed with Adobe Photoshop 6.0 (Adobe Systems, San Jose, CA).

Numerical Expression of Cell Attachment

Panels for measuring relative attachment preferences were established from cell clusters at randomly selected levels. The circumference of a specific cell type in a cluster, and the length of attaching lines to counter-immunostained cells were determined by use of the computer software. Relative attachment preference was defined as the rate of cell attachment, using the following formula:

  • equation image

Data Analysis

Cultured cells from three or four animals in each experiment were statistically analyzed. Clusters were randomly selected under scanning microscopy to measure the rates of cell attachment. Profiles of immunostained GH (n = 922), PRL (n = 814), ACTH (n = 314), TSH (n = 237), and LH cells (n = 343) were obtained from over 30 clusters. The rates of attachment of one type of cell to other types are difficult to compare because the varying numbers and shapes among cell types directly affect attachment preferences. Therefore, we normalized the attachment rates of various types of cells to one specific type of cells in a panel to eliminate this problem and to generate comparable values when the relative attachment preference was not specific. The attachment preferences of hormone-producing cells are presented as means + S.E.M. They were statistically analyzed by use of the nonparametric Kruskal-Wallis analysis of variance (ANOVA) of ranks, followed by a test for outliers.

Electron Microscopy Observation

For observation of junctional complexes in clusters, cultured cells were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2 for 30 min at 4°C. After the cells were washed repeatedly with 0.1 M cacodylate buffer, they were postfixed with 1% OsO4 in 0.1 M cacodylate buffer for 30 min at 4°C, dehydrated in a series of graded alcohols, and embedded in epoxy resin (Quetol 812; Nissin EM Co., Tokyo, Japan). The cells were sectioned into ultrathin slices with a Reichert-Nissei Ultracut S (Leica Inc., New York, NY), stained with uranyl acetate and lead citrate, and then observed under a Hitachi H-7600 electron microscope.

RESULTS

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

Microscopy Observation of Topographic Cell Affinity

After the anterior pituitary cells were cultured for 72 hr, they assumed solitary, paired, or clustered states. Most clusters comprised less than a dozen cells. Figure 1 shows cluster formation by pituitary cells. As the cells settled to the surface of the culture dishes, they appeared to form cell aggregates without migration in 1 hr. The aggregates gradually compacted to form clusters, and thereafter the number of cells remained constant. To determine whether cells in clusters proliferate, we used immunocytochemistry with a mouse anti-PCNA monoclonal antibody (ICN Biomedicals Inc., OH). All of the cells were negative for anti-PCNA antibody, indicating that cluster formation is not related to cell proliferation (data not shown). All five types of hormone-producing cells (GH, PRL, ACTH, TSH, and LH) formed clusters. With the exception of the PRL cells, the morphology of the cells did not significantly differ from that in vivo. The PRL cells were rounder and larger than those in vivo. We used confocal laser microscopy to determine how the cell types formed clusters. Figures 2–4 are extracted two-dimensional images that show the topographic affinities of all combinations of the hormone-producing cells. The following is a summary of our findings based on observations of hundreds of cells of each type. Round GH cells attached to all types of cells, but more frequently to GH cells (Fig. 2). Figure 2 is a typical image of a GH cell surrounded by an ACTH cell in a cluster. The topographic relationship between round PRL cells and LH cells was specific (Fig. 3). Some PRL cells entirely enveloped an LH cell. Other PRL cells attached to ACTH or TSH cells, but not as frequently as to LH cells. ACTH cells were stellate, polygonal, or irregularly shaped, and frequently enveloped GH cells (partially or entirely) with their cell projections (Fig. 2). ACTH cells seldom attached to ACTH, TSH, or LH cells (Fig. 4). TSH cells assumed various shapes (round or oval, stellate, polygonal, or irregular) and mainly attached to GH and PRL cells. LH cells, which were always round or oval, frequently attached to GH or PRL cells (Fig. 3). Two or three PRL cells often surrounded a single LH cell.

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Figure 1. Phase contrast microscopy observation of cluster formation. Cells in culture were video-recorded at a fixed point under phase contrast microscopy. a-h: Extracted images acquired 0, 5, 10, 20, and 30 min; and 1, 3, and 24 hr, respectively, after the cells were plated. Bar: 50 μm.

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Figure 2. Immunofluorescence-stained GH (green) and ACTH cells (red) in primary monolayer culture. Polygonal ACTH cells enclose a GH cell via cell projections. One ACTH cell totally envelopes a GH cell (arrow). Bar: 50 μm.

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Figure 3. Immunofluorescence-stained PRL (green) and LH cells (red) in primary monolayer culture. An LH cell is enveloped by two or three PRL cells (arrow). Bar: 50 μm.

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Figure 4. Immunofluorescence-stained ACTH (green) and LH cells (red) in primary monolayer culture. Bar: 50 μm.

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Statistical Analysis of Topographic Cell Affinity

The rates of attachment between the types of hormone-producing cells were statistically analyzed using a computer program, and the results are shown in Figs. 5 and 6. According to the Kruskal-Wallis test, the rates of attachment of the five types of cells to GH, PRL, and LH cells (Fig. 5a–c) were significantly different, indicating that there were specific topographic affinities among cell types. The rates of attachment of ACTH to GH cells (Fig. 5a), LH to PRL cells (Fig. 5b), and PRL to LH cells (Fig. 5c) were significantly high, and that of PRL to PRL cells was significantly low (Fig. 5b). This indicates close or remote arrangements, respectively, among the cell types. The values in Fig. 6 were not analyzed, since significant heterogeneities were absent (as described above). We investigated whether the values of these 25 combinations in vitro (Figs. 5 and 6) correlated with the in vivo values found in our previous study (Figs. 9-13 in Noda et al., 2001). Statistical analyses showed a significant overall correlation between the rates in vivo and those in vitro (P < 0.05, r = 0.67; Pearson's correlation coefficient test; Fig. 7).

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Figure 5. Rates of attachment to (a) GH, (b) PRL, and (c) LH cells. The average percentage of the circumference of all cell types attached to (a) GH, (b) PRL, and (c) LH cells is graphed. Vertical lines represent the S.E.M. The rates of cell attachment of five types of cells to each type are significantly different among types (Kruskal-Wallis test, P < 0.01). Values of (a) ACTH cells to GH cells, (b) LH cells to PRL cells, and (c) PRL cells to LH cells were significantly higher (P < 0.01, asterisk), and that of PRL cells was significantly lower (P < 0.01, asterisk) among five tested groups.

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Figure 6. Rates of attachment to (a) ACTH and (b) TSH cells. The average percentage of the circumference of all cell types attached to (a) ACTH and (b) TSH cells is graphed. Vertical lines represent the S.E.M. The Kruskal-Wallis test revealed no significant differences among types.

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Figure 9. Electron micrograph of a cluster primarily cultured for 72 hr. An ACTH cell, characterized by the row arrangement of secretory granules along the cell membrane, encircles a neighboring GH cell. G, GH cell; A, ACTH cell. Bar: 10 μm. Inset shows the region outlined by the rectangle at a higher magnification. No junctional complexes are evident between these cells.

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Figure 7. Correlation between rates of cell attachment in vivo and in vitro (n = 25). Statistical analysis showed a significant positive correlation between rates in vivo (Figs. 9–13 in Noda et al., 2001) and those in vitro (P < 0.05, r = 0.671; Pearson's correlation coefficient test).

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Electron Microscopy Observation of the Clusters

To determine whether there were junctional complexes between the cells, we observed the clusters by electron microscopy. The cultured cells formed clusters connected by cell bodies or projections (Figs. 8 and 9). Some intermediate junctions and desmosomes were identified between the F-S cells, especially surrounding the folliculo-lumen (Fig. 8). On the other hand, junctional complexes including gap junctions were scarce between hormone-producing cells, as well as between hormone-producing and F-S cells, among hundreds of clusters (Fig. 9).

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Figure 8. Electron micrograph of a cluster primarily cultured for 72 hr. In addition to hormone-producing cells, several dispersed F-S cells (non-hormone-producing cells) gathered to form a pseudolumen within the central portion. Microvilli are observed in the lumen. A desmosome (arrow) and long intermediate junctions (double arrow) can be seen between F-S cells. * indicates the outside of the cell cluster. Bar: 1 μm.

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DISCUSSION

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

Previous electron microscopy studies have shown that various hormone-producing cells in the rat anterior pituitary have specific affinities for each other (Nakane, 1970; Siperstein and Miller, 1970; Yashiro et al., 1981; Yoshimura and Yogami, 1981; Nogami and Yoshimura, 1982; Shirasawa et al., 1985). In an earlier work we numerically expressed topographic cell affinity from data acquired using confocal laser microscopy (Noda et al., 2001). Furthermore, our statistical analysis objectively confirmed that some combinations (such as GH and ACTH cells) locate in juxtaposition (Nakane, 1970; Siperstein and Miller, 1970; Yoshimura and Nogami, 1981), and that PRL and LH/FSH cells (Nakane, 1970; Nogami and Yoshimura, 1982) have a high affinity for each other. Conversely, cells that do not attach (such as ACTH and TSH types (Nakane, 1970)) have a specific and low affinity for each other.

Anterior pituitary cells in primary culture tend to form clusters (Fig. 1). Our results indicate that cluster formation issues from contingent encounters and circumstantial attachment of the cells—not from active migration. Furthermore, cells in clusters were immunohistochemically negative for anti-PCNA antibody, which suggests that cluster formation is caused only by cell attachment. A few investigators previously described the topographic affinity between clustered cells. Allaerts et al. (1991) studied the topographic affinity of hormone-producing cells in vitro and found that enriched suspension cultures of PRL and LH cells have an affinity for each other. Wilfinger et al. (1984) showed that LH cells position themselves in close proximity to PRL cells after they are plated at high density, which enhances cell-to-cell interaction in clusters. However, none of these reports examined all combinations of known hormone-producing cells. The present work is the first to describe the ability of anterior pituitary cells to reconstruct specific cell attachments in primary monolayer culture.

The morphological features of clustered hormone-producing cell types in vitro are essentially identical to those in vivo, with the exception of PRL cells, which are larger and mainly oval in vitro (Fig. 3) but polygonal or irregularly shaped in vivo (Nogami and Yoshimura, 1982; Shirasawa et al., 1985). Wilfinger et al. (1984) and Snyder et al. (1976) reported that PRL cells changed round shape in vitro, and Orgnero de Gaisán et al. (1997) suggested that these morphological changes are the result of the disruption of the normal paracrine relationship and hypothalamic regulatory system. Our observations showed that the shape of the PRL cells changed in vitro, but the number did not increase. Characteristic cell attachment in vivo between specific cell types was notably reconstructed in vitro (Figs. 2–4). For instance, the attachment of ACTH to GH cells via cell projections (Fig. 2), and the envelopment of an LH cell by two or three PRL cells (Fig. 3) were similar to our previous findings in vivo (Noda et al., 2001).

Statistical analyses of numerically expressed cell affinities showed that the rates of cell attachment varied according to the combination of cell types (Figs. 5 and 6). The rate of cell attachment should vary with the degree of specificity of their topographic cell affinities. If none of these cells had specific affinities, the rates among cell types would be similar. Our statistical analyses showed that cell type topographic affinities are specific in vitro, at least with some combinations of cell types (Fig. 5a–c). However, it is difficult to compare rates among cell types because the numbers and shapes of the cells directly affect the rates. We therefore compared the rates of attachment of various types of cells to one other type of cell.

Our previous study (Noda et al., 2001) showed that the affinities of hormone-producing cells are specific in vivo. We examined the correlation between the rates of cell attachment in vitro and the corresponding values in vivo (n = 25; Figs. 9–13 in Noda et al., 2001). Statistical analyses showed a significant overall correlation between the rates in vivo and those in vitro (Fig. 7). This finding suggests that the specific arrangement in vivo was at least partly reconstructed in vitro. Combinations with specific high or low affinities were essentially the same in vivo and in vitro. These findings indicate that the hormone-producing cells of the rat anterior pituitary can reconstruct specific topographic affinities in vitro.

Our observations revealed that the cells formed clusters attached to each other at random, and that the clusters became smaller without a decrease in the number of cells (Fig. 1). Therefore, topographic affinity may be reconstructed after cluster formation is mediated by unknown factors, such as junctional complex and cell adhesion molecules. We observed intermediate junctions and desmosomes between F-S cells (Fig. 8), but very few junctions between hormone-producing cells despite high-affinity pairing (GH and ACTH cells in Fig. 9). These findings are mostly consistent with previous in vivo reports that showed tight junctions, intermediate junctions, and desmosomes (Soji and Herbert, 1989) between F-S cells, but only desmosomes between PRL cells (Saunders et al., 1982). In vitro studies have found tight junctions and gap junctions in aggregates (Wilfinger et al., 1984), tight junctions and intermediate junctions between F-S cells (Allaerts and Denef, 1989), and tight junctions in PRL cell/GH cell-enriched and LH cell-enriched aggregates (Van der Schueren et al., 1982). However, these investigations were performed under specific conditions; some types of cells were concentrated, and the topographic affinity in vivo was ignored. The scarcity of junctional complexes, at least in our culture system, indicates that they are not the cause of reconstructed topographic affinity between hormone-producing cells. Some cell adhesion molecules have been identified in the rat anterior pituitary, including NCAM (Langley et al., 1987; Berardi et al., 1995) and PB-cadherin (Sugimoto et al., 1996). Spangler and Delidow (1998) observed P- and N-cadherin expression in PRL-producing rat pituitary 253-1 cells, and Heinrich et al. (1999) reported that N-cadherin mediates cell aggregation in rat somatolactotropic GH3 cells. These molecules may serve a function during cluster formation, but there is no evidence that cell adhesion molecules play a role in topographic affinity in the anterior pituitary. Some suggestions have been proposed on the role of cell adhesion molecules in organization of other endocrine tissues, such as the pancreatic islets, that have various types of hormone-producing cells. Some distribution patterns (such as that of B cells concentrated in the center of the islets, and of A, D, and PP cells dispersed around B cell clusters) can be reconstructed according to the expression of NCAM or cadherin in vitro (Cirulli et al., 1994). Cirulli et al. (1994) suggested that these differences in expression among cell types contribute to the characteristic distribution of cells within the islets of Langerhans. Because the topographic affinity in Langerhans islets tends to be between homotypical cells, and cadherins mainly mediate attachment between them, heterotypical topographic affinity in the anterior pituitary might not be caused solely by cadherins, but by some other, unknown factors as well. This issue should be further investigated.

The functional significance of specific pairing between hormone-producing cells remains unclear. The attachment of one cell to another is convenient for direct cell-to-cell communication, but we did not observe gap junctions between cultured hormone-producing cells in this study. Intracellular paracrine communication does not always require communicating cells to be juxtaposed. However, a report by Abraham et al. (1996) showed that PRL gene expression differs depending on whether a mammotrope is alone, is in contact with one other mammotrope, or adheres to a non-mammotrope.

In conclusion, cultured rat anterior pituitary hormone-producing cells form clusters with a specific cell-to-cell affinity similar to that in vivo. Some cell adhesion molecules may be involved with cluster formation. The present study provides information that will be helpful in future investigations of intracellular communication.

Acknowledgements

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

We are grateful to K. Inose and M. Yatabe for their excellent technical assistance. This study was partly supported by “The Research Award to JMS Graduate Student” from Jichi Medical School.

LITERATURE CITED

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