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Max-Planck-Institut für experimentelle Endokrinologie, D-30603 Hannover, Germany (A.P., H.H., L.S., K.B.); Department of Internal Medicine III and Clinical Endocrinology (W.J.d.G.) and Department of Endocrinology and Reproduction (T.J.V.), Faculty of Medicine and Health Sciences, Erasmus University, Rotterdam, The Netherlands 3000DR
Address all correspondence and requests for reprints to: Prof. Dr. Karl Bauer, Max-Planck-Institut für experimentelle Endokrinologie, P.O.B. 610309, D-30603 Hannover, Germany. E-mail: 106001.2503{at}compuserve.com
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Abstract |
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When AP cells were cultured as reaggregates in the same (FCS-containing) medium, only a marginal increase in TRH content and preproTRHmRNA levels was observed. Irrespective of the culture systems and the culture conditions used, TRH gene expression was not observed when FCS was omitted. These results indicate that TRH gene expression more likely reflects derepression, rather than induction, of the TRH gene.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Based on these findings, it has been suggested that TRH may not only control the function of the AP in an endocrine fashion via release into the hypophyseal portal blood system, but also in a paracrine or autocrine manner through local synthesis and release (7, 10). However, the mechanisms of TRH gene expression have not been elucidated yet. Because signaling through paracrine regulators depends on the local cell-to-cell communication systems (for reviews, see Refs. 10, 11, 12, 13, 14, 15), we were interested in studying the biosynthesis and release of TRH by cultivating AP cells, either as monolayers or as reaggregate cultures, under various conditions.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rat AP cell culture
Immediately after decapitation, APs were removed aseptically and
minced with a razor blade. Tissue blocks were treated enzymatically
using trypsin and were finally dispersed mechanically to single cells,
as described by Denef et al. (16, 17). Cells were either
plated on 35-mm petri dishes (coated with poly-D-lysine;
plating density 
1250 cells/mm2) or they were
allowed to reassociate into three-dimensional cell reaggregates by
continuous gyratory shaking. AP cells were cultured for up to 21 days
in a humified CO2-incubator at 37 C. The chemically defined
culture medium consisted of DMEM/Ham’s F12 1:1 medium, supplemented
with 1 g/liter NaHCO3, BSA, transferrin, insulin,
ethanolamine, sodium selenite, ethanol, catalase, penicillin, and
streptomycin (16). As indicated, heat-inactivated FCS (10%
vol/vol) (Cytogen GmbH, Lohmar, Germany) was added to the culture
medium at day 2 of culture. Every second day, half of the medium was
replaced by fresh medium.
Extraction procedures
For TRH measurements, AP, neurointermediate lobe, and
hypothalamic tissue were collected. The hypothalamus was removed, first
by freeing the sides of its lateral borders, and then dissecting a 2
mm-deep piece of tissue anteriorly bordered by the optic chiasm and
posteriorly by the anterior margin of the mammillary nucleus. Rat
tissue, cultivated pituitary monolayer cells, or reaggregates were
homogenized in 70% methanol/30% 2 N acetic acid. After
sonication and centrifugation at 10,000 x g for 10
min, the supernatants were lyophilized and reconstituted for RIAs in
0.5 M sodium phosphate buffer (pH 7.4). DNA was determined,
according to Downs and Wilfinger (18), using the fluorescent dye
bisbenzimidazol and calf thymus DNA as standard. Protein was measured
by the method of Peterson using serum albumin as standard (19).
RIAs
TRH-immunoreactive material (TRH-IR) was determined by RIA using
either the TRH-specific antiserum no. 8880 at a final dilution of
1:10,000 or the less-specific antiserum no. 4319 at a final dilution of
1:2500. Both antisera have been extensively characterized, as described
previously (20, 21). TRH was iodinated by the chloramine T method, and
the monoiodinated TRH was isolated by TLC, as described by Grouselle
et al. (22). The samples (0.3 ml) were incubated at 4 C for
48 h, and then 0.1 ml pig IgG (2.7 mg protein/ml) and 0.5 ml 40%
polyethylene glycol was added to precipitate bound TRH. After
centrifugation, the radioactivity of the pellets was determined by use
of a 
counter (1470 Wizard, Wallac, Turku, Finland). The detection
limit was 1–2 pg/tube (no. 4319) or 3–5 pg/tube (no. 8880). All
results are presented as TRH equivalents.
Northern blot analysis
AP cells (2, 4 mg protein) were homogenized in 4 ml
SDS-containing Tris-buffer (0.1 M Tris, 0.5 M
LiCl, 10 mM EDTA, 5 mM dithiothreitol, 1% SDS,
pH 8). Polyadenylated [poly(A)+]-enriched RNA was
isolated directly from the homogenates using magnetic
oligo(deoxythymidine)25 polystyrene beads (Deutsche Dynal,
Hamburg, Germany), according to the manufacturer’s instructions.
Poly(A)+enriched RNA (10 µg/lane) was separated by electrophoresis in denaturing agarose gels (2.2 M formaldehyde and 1.5% agarose), capillary transferred to nylon membranes (Nytran NY 12 N, Schleicher and Schuell, Dassel, Germany), and cross-linked by UV irradiation.
Hybridizations were performed under high-stringency conditions (42 C, 16 h in 50% formamide, 0.5% SDS, 100 µg/ml salmon DNA, 0.5 M NaCl, 12 mM EDTA, and 0.09 M sodium phosphate, pH 7.4) with 50-ng complementary DNA (cDNA) fragments randomly labeled with [32P]deoxy-CTP to high specific activities. The following cDNA fragments were used as probes: a 0.8-kb fragment of the rat cDNA encoding preproTRH (23); a 0.7-kb fragment of the rat cDNA encoding PRL (24); and, as standard, a 1.1-kb fragment of human cDNA encoding glyceraldehyde-3-phosphate dehydrogenase, obtained from Clontech (ITC Biotechnology, Heidelberg, Germany).
Release experiments
The cells were washed twice with standard buffer, pH 7.4,
consisting of 20 mM HEPES, 129 mM NaCl, 5
mM KCl, 1.2 mM CaCl2, 1.2
mM MgSO4, 1 mM
Na2HPO4, and 10 mM glucose. The
cells were incubated in this buffer for 30 min to determine basal TRH
release and were then exposed for 30 min either to the same buffer
(containing, however, 55 mM KCl and 79 mM NaCl)
or to the standard buffer supplemented with the calcium ionophore A
23187 (10 µM; Sigma, Deisenhofen, Germany), the releasing
factors GnRH (100 nM; Bachem, Heidelberg, Germany), CRH (10
nM; Saxon Biochemicals, Hannover, Germany), and GRF (10
nM; Saxon Biochemicals), respectively. The cells were then
incubated for 30 min again in the standard buffer. For RIA
measurements, the cells were extracted, as described above, and
aliquots of the incubation media were acidified with 2N acetic acid and
then lyophilized.
In situ hybridization (ISH) and immunocytochemistry (ICC)
A 0.8-kb cDNA fragment, corresponding to the entire coding
region of the rat cDNA encoding preproTRH (23), was inserted in the
EcoRV-site of pBS KS II+ (Stratagene, Heidelberg, Germany)
and linearized with HindIII (for the synthesis of sense
riboprobe) or PstI (for the synthesis of antisense
riboprobe). Digoxigenin (dig)-11-UTP-labeled cRNA probes were generated
from the template using a RNA transcription kit (Boehringer Mannheim,
Mannheim, Germany) with T3 (antisense) or T7 (sense) RNA
polymerase.
Cells plated on 3-aminopropyltriethoxy silane-coated glass slides were washed with PBS (0.01 M phosphate buffer, 0.15 M NaCl, pH 7.2) and fixed with 4% paraformaldehyde in PBS for 5 min. The cells were treated consecutively with 0.1% Triton-X-100 in PBS for 3 min, followed by PBS and 0.25% vol/vol acetic anhydride in 0.1 M triethanolamine, pH 8, and 0.5% vol/vol acetic anhydride in the same buffer for 5 min at room temperature. Cells were then rinsed in 2 x saline-sodium citrate (SSC) (1 x SSC = 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0) and air-dried. Hybridization was carried out in a solution containing 40% formamide, 4 x SSC, 10% dextran sulfate, 1 x Denhardt’s solution (Ficoll 400, polyvinylpyrrolidone, BSA, each 0.02%), 0.1 M dithiothreitol, 100 µg/ml denatured salmon sperm DNA, 100 µg/ml Escherichia coli transfer RNA, and 50–200 ng dig-labeled cRNA-probe. Slides were incubated overnight at 50 C in a moist chamber. After the hybridization, slides were washed in 2 x SSC and treated with ribonuclease A (10 µg/ml; Sigma) at 37 C for 30 min. Slides were successively washed in 2 x, 1 x, 0.5 x, and 0.1 x SSC for 10 min each at room temperature and then in 0.1 x SSC for 30 min at 60 C. After this procedure, the slides were exposed to alkaline phosphatase conjugated antidig antibody (Boehringer Mannheim, 1:500 diluted) for 3 h at room temperature, followed by overnight incubation in chromogen solution containing nitroblue tetrazolium (NBT; 0.41 mM) and 5-bromo-4-chloro-3-indolylphosphate (0.38 mM). The color reaction was stopped by rinsing the slides in TE-buffer (10 mM Tris • HCl, 1 mM EDTA, pH 8) for 10 min.
After ISH, the cells were washed with PBS and were incubated with 0.2 ml guinea pig LH-ß antiserum (lot no. AFP 22238789, dilution 1:2000, a gift from Dr. A. F. Parlow, NIADDK) for 14 h in a humid chamber at 4 C. After several washes with PBS, the cells were incubated for 30 min at room temperature with 0.2 ml Cy3-conjugated goat antiguinea pig antibody (dilution 1:200, Dianova, Hamburg, Germany), rinsed with PBS, covered with mounting medium (Citifluor, Plano, Marburg, Germany) to retard fading, and examined under a fluorescence microscope (Olympus BH3, New York, NY).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
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TRH synthesis by monolayer and reaggregate cell cultures
As shown in Fig. 1, TRH content exponentially
increased with time when AP cells were kept as monolayer cultures in
medium containing 10% FCS. In contrast, when AP cells were cultured as
reaggregates under otherwise identical conditions, only a marginal
increase in TRH content (10.5 ± 1.5 pg/µg DNA, compared with
188 ± 8.5 pg/µg DNA in monolayer cultures) could be detected
after 3 weeks in culture. Regardless of the culture system employed,
synthesis of TRH was completely dependent on the addition of FCS.
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). PreproTRHmRNA was not detectable when the cells were
kept in FCS-free medium, a weak signal was detected when reaggregates
were kept in FCS-containing medium, and a dramatic increase in the
transcript level was found when the cells were kept as monolayer
cultures in FCS-containing medium for 3 weeks. Because fibroblasts
under the latter conditions are known to increase considerably with
time, both TRH-content and the mRNA levels are significantly
underestimated.
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). Among the
hypothalamic releasing factors tested (CRH; GRF and GnRH) only GnRH
evoked a significant secretion of TRH, indicating that TRH may be
stored within gonadotropes.
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) supported the findings of the release
experiments indicating that preproTRHmRNA was contained in gonadotropes
or, at least, in a subpopulation of these cells. At present, we cannot
exclude the possibility that preproTRHmRNA also is expressed in other
cell types at a lower level.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As a putative hormone and signaling molecule, TRH should be contained in a releasable pool. Indeed, release could be evoked by high K+, as described by Bruhn et al. (4). Release of TRH was stimulated also by the Ca++ ionophore A23187, as well as by GnRH, but not by CRH or GRF, suggesting that TRH is synthesized by gonadotropes. In agreement with this notion, colocalization of preproTRHmRNA and LH could be demonstrated at the light microscopic level by a combination of ISH and ICC. This finding correlates well with the cytochemical studies of Childs and co-workers who localized TRH in secretory granules of gonadotropes and corticotrope-like cells after long-term culture (3), whereas Bruhn et al. (7) observed high expression of the TRH gene within a subpopulation of somatotropes. These differences might be caused by subtle differences in the experimental conditions (e.g. the composition of the FCS used) and might not be too surprising, especially when taking into account that the cells were cultured under artificial conditions for long periods of time.
Because we were interested in studying the regulation of TRH biosynthesis in APs, we also cultured the cells in serum-free, chemically defined medium that is generally used for culturing pituitary cells (16, 17). As reaggregates, pituitary cells can be cultured in this medium for several months without significant changes in morphology or function (16, 17). In serum-free medium, synthesis of TRH or TRH-like peptides could be observed neither in monolayer nor in reaggregate cultures.
Previous studies by Bruhn et al. (8, 9) clearly demonstrated that TRH gene expression in AP cell cultures is strongly stimulated by thyroid hormones and potentiated by glucocorticoids, whereas TRH gene expression in hypothalamic neurons in vivo is strongly inhibited by thyroid hormones (26, 27). In serum-free, as well as in FCS-containing medium, neither T3, nor dexamethasone, nor the combination of these 2 hormones had any effect on the expression of the TRH gene under our experimental conditions. These discrepancies may be caused by the experimental protocol, considering that charcoal-stripped FCS or FCS subjected to the ion exchange resin AG 1-X8 were used by Bruhn and co-workers (8, 9).
In our cultures, synthesis of TRH was observed only when the cells were cultured in the presence of FCS, which could be replaced by new-born calf serum but not by calf serum, bovine serum, or horse serum. Synthesis of TRH also was not observed in our culture systems when the serum-free medium was supplemented by various growth factors, suggesting that yet unidentified growth factors and/or differentiation factors are present in FCS that stimulate either directly or indirectly (via stimulation of other cells) the synthesis of TRH in long-term monolayer cultures.
However, TRH synthesis seems not to be determined by such serum factors alone, because radioimmunassayable levels of authentic TRH could not be detected in rat APs, regardless of the donor age (4). Because cell-cell interactions are well known to play a fundamental role in developmental processes and gene expression (28, 29, 30), we were interested in comparing the biosynthesis of TRH by the monolayer cell cultures with that of reaggregate cultures. Extensive studies by Denef and co-workers (16, 17) demonstrated that pituitary cells do not associate randomly but form three-dimensional structures that become organized within a few days in a tissue-like configuration (17). Thus, these structures can be used as an ideal in vitro model to study paracrine interactions in the AP. Interestingly, compared with the monolayer cultures, TRH gene expression in aggregate cultures was strongly reduced, even when the cells were cultured for 3 weeks in medium containing 10% FCS. This result seems to indicate that TRH synthesis by the monolayer cultures reflects derepression, rather than induction, of the TRH gene. If this interpretation is correct, it is not surprising that authentic TRH is almost absent in the intact AP of adult, as well as neonatal, animals. Whether authentic TRH is synthesized under certain pathological conditions, as indicated by some reports (5, 6), remains to be investigated further.
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Acknowledgments |
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Footnotes |
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Received February 7, 1997.
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References |
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. A model for membrane-anchored growth factors. J Biol
Chem 265:21393–21396This article has been cited by other articles:
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  J. Schwartz Intercellular Communication in the Anterior Pituitary Endocr. Rev., October 1, 2000; 21(5): 488 - 513. [Abstract] [Full Text]
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 J. Ehrchen, A. Peters, D. K. Lüdecke, T. Visser, and K. Bauer Analysis of Thyrotropin-Releasing Hormone-Signaling Components in Pituitary Adenomas of Patients with Acromegaly J. Clin. Endocrinol. Metab., August 1, 2000; 85(8): 2709 - 2713. [Abstract] [Full Text]
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J. J. Evans Modulation of Gonadotropin Levels by Peptides Acting at the Anterior Pituitary Gland Endocr. Rev., February 1, 1999; 20(1): 46 - 67. [Abstract] [Full Text]
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, •) or as
reaggregates (
, 
) in chemically defined culture medium,
supplemented (
4, values are means ±
SD). *, P < 0.05; **,
P < 0.01 (control vs. incubation
with secretagogues).