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Rat anterior pituitary cells in vitro can partly reconstruct in vivo topographic affinities.

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THE ANATOMICAL RECORD PART A 272A:548 –555 (2003)
Rat Anterior Pituitary Cells In Vitro
Can Partly Reconstruct In Vivo
Topographic Affinities
TAKAHIRO NODA, MOTOSHI KIKUCHI, SACHIKO KAIDZU, AND
TAKASHI YASHIRO*
Division of Histology and Cell Biology, Department of Anatomy, Jichi Medical
School, Tochigi, Japan
ABSTRACT
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 hormoneproducing 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.
Key words: anterior pituitary gland; topographic cell affinity; primary monolayer culture; immunohistochemistry; confocal laser microscopy
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) pub©
2003 WILEY-LISS, INC.
lished 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 fre-
Grant sponsor: Jichi Medical School, “The Research Award to
JMS Graduate Student”.
*Correspondence to: Takashi Yashiro, Division of Histology
and Cell Biology, Department of Anatomy, Jichi Medical School,
3311-1 Yakushiji, Minamikawachi, Tochigi 3290498, Japan. Fax:
⫹81-285-445243. E-mail: tyashiro@jichi.ac.jp
Received 5 December 2002; Accepted 12 February 2003
DOI 10.1002/ar.a.10065
AFFINITIES AMONG PITUITARY CELLS IN VITRO
quently 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
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 ␮g/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 ␮l 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.,
549
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 ␤ 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 ␤ subunit (1:3,200) and guinea pig antibody against rat LH ␤ 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
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NODA ET AL.
Fig. 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 ␮m.
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:
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.
rate of cell attachment (%)
⫽ length of line attached to counterstained cells/
circumference of stained cells ⫻ 100
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
RESULTS
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
AFFINITIES AMONG PITUITARY CELLS IN VITRO
551
Fig. 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 ␮m.
Fig. 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 ␮m.
Fig. 4. Immunofluorescence-stained ACTH (green) and LH cells
(red) in primary monolayer culture. Bar: 50 ␮m.
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.
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).
Statistical Analysis of Topographic Cell Affinity
Electron Microscopy Observation of the
Clusters
The rates of attachment between the types of hormoneproducing 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
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).
552
NODA ET AL.
Fig. 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.
Fig. 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.
DISCUSSION
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
AFFINITIES AMONG PITUITARY CELLS IN VITRO
Fig. 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).
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
553
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
554
NODA ET AL.
Fig. 8. Electron micrograph of a cluster primarily cultured for 72 hr. In
addition to hormone-producing cells, several dispersed F-S cells (nonhormone-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 ␮m.
Fig. 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 ␮m. Inset shows the region outlined by the
rectangle at a higher magnification. No junctional complexes are evident
between these cells.
AFFINITIES AMONG PITUITARY CELLS IN VITRO
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-tocell 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 hormoneproducing 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.
ACKNOWLEDGMENTS
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.
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