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Department of Poultry Science and Center for Animal Biotechnology (T.E.P.), Institute of Biosciences and Technology, Texas A&M University, College Station, Texas 77843-2472
Address all correspondence and requests for reprints to: Dr. T. E. Porter, Department of Animal and Avian Sciences, 4111 Animal Sciences Center, University of Maryland, College Park, Maryland 20742-2311.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell cultures
Cells were cultured according to the procedure described
previously (7). Recovered anterior pituitary cells were plated
(2.0 x 105 cells/well) in
poly-L-lysine-coated 12-well tissue culture plates. Cells
were allowed to attach for 45 min, and wells were then filled (2 ml)
with serum-free medium. The medium consisted of a 1:1 mixture of phenol
red-free medium 199 and Ham’s F-12 nutrient mixture supplemented with
0.1% BSA, 5 µg/ml human transferrin, 5 µg/ml bovine insulin, 100
U/ml penicillin G, and 100 µg/ml streptomycin sulfate. Cells were
cultured for 2 days in a humidified tissue culture incubator (37.5 C;
5% CO2) and then harvested for detection of GH-secreting
cells by reverse hemolytic plaque assays. Approximately 80% of all
cells plated were harvested after culture, and no differences were
observed between treatments in the numbers of cells recovered
throughout this study. The GH content of the culture medium was
assessed by RIA (8). All samples were assayed in a single RIA, and the
intraassay coefficient of variance was 8.3%. The sensitivity of this
assay was 2.5 ng/ml.
Reverse hemolytic plaque assay (RHPA)
The RHPA procedure, which allows detection of hormonal secretion
by individual cells, was originally described by Neill and Frawley (9).
The assays were performed according to the protocol described in detail
previously (10), using rabbit antiserum against chicken GH and
modifications described earlier (6). Briefly, pituitary cells (1.0
x 105/ml) were mixed with an equal volume of an 18%
suspension of protein A-coated ovine erythrocytes and infused by
capillary action into previously constructed Cunningham chambers. Cells
were allowed to attach for 45 min (37.5 C; 95% air and 5%
CO2), then chambers were rinsed with DMEM to remove
unattached cells. DMEM containing GH antiserum (1:40) with or without
human GH-releasing hormone (GHRH; 1.0 x 10-7
M), was then added to the resulting monolayer of cells, and
replicate chambers were incubated for 8 or 20 h (three chambers
each). Plaque formation was induced by a 45-min incubation with guinea
pig serum as a source of complement (1:80). The cells were then fixed
with 2% glutaraldehyde in 0.9% saline and stained with methyl green.
The proportions of plaque formers were determined by evaluation under a
light microscope, with a minimum of 100 pituitary cells counted per
chamber.
Biochemical characterization
Column chromatography.
Three samples of lyophilized e-16
serum (150 µl each) were dissolved in 1 M acetic acid for
30 min at room temperature. Samples were then centrifuged to remove
undissolved material, and the supernates were applied to a Sephadex
G-50 column, (32 x 1.6 cm; equilibrated with 1 M
acetic acid). Twenty-five fractions (3 ml each) were collected and
lyophilized. Each fraction was dissolved in 3 ml water and lyophilized
again to remove any residual acetic acid. The fractions were then
dissolved in 3 ml PBS (pH 7.4, 0.04 M) and tested for their
ability to stimulate somatotroph differentiation in cultures of e-12
pituitary cells (bioassay). Relative molecular mass was determined by
elution of protein standards. First, fractions were combined into three
pools: void volume (>25 kDa; pool I), fractions between void volume
and salt fraction (25–1.5 kDa; pool II), and salt fraction (<1.5 kDa;
pool III). Each pool was tested for bioactivity. The fractions of the
pool that showed bioactivity were then tested individually. Volumes of
individual fractions were selected to yield an effective concentration
of 1% serum in culture based on the assumption that all of the
bioactivity was contained in a single fraction or a pool of
fractions.
Proteolytic hydrolysis.
Serum samples from e-12 and e-16
(three of each age) were subjected to enzymatic digestion with trypsin
conjugated to agarose beads (Sigma) for 1 h in PBS (1.8 U; 37.0 C;
pH 7.4). After digestion, the trypsin-coated beads were removed by
centrifugation, and the supernates were applied to day 12 pituitary
cells in culture for bioassay. As controls, PBS alone and after
incubation with the enzyme was bioassayed as well.
Ether extraction.
Serum samples from e-12 and e-16 (three of
each age) were first heated to 70 C for 1 h and then extracted
twice with 5 vol ethyl ether. Samples that were not treated and samples
that were heated but not extracted were included as controls. All
samples were lyophilized and resuspended in PBS, then samples (1% by
volume) were applied to day 12 pituitary cells in culture for
bioassay.
Steroids and steroid receptor antagonists
All steroids and steroid receptor antagonists were dissolved in
100% ethanol initially (1 x 10-3 M).
Further dilutions were made in PBS. Each steroid was tested for
bioactivity at different concentrations
(10-12–10-7 M). The final
concentration of ethanol was consistent across all wells (0.1%),
including control wells.
Immunoprecipitation
Rabbit anticorticosterone and rabbit anti-GH were tested for
their ability to block serum-induced somatotroph differentiation
in vitro. Treatments consisted of heated e-16 serum and
corticosterone. Rabbit anti-GH serum was included as a control for the
endogenous effects of serum. Each of the treatments and antisera were
also bioassayed alone as controls. The GH antiserum control was that
used for the RHPA (6). The corticosterone antiserum was obtained from
Fitzgerald (Concord, MA). Its cross-reactivity is less than 0.3% with
cortisol and 0.01% with progesterone and other steroids. The antisera
(10 µl diluted 1:5 with PBS/treatment) were first incubated (1 h,
37.0 C) with protein A-bound to acrylic beads (Sigma) to eliminate
steroids and other components in the antisera and to test the IgG
fraction alone. After incubation, the tubes were centrifuged,
supernatants were removed, and the IgG-protein A-bead complexes
were rinsed with equal volumes of PBS. Rinsing was repeated five times.
Steroid or embryonic serum treatments were then added to the
IgG-protein A beads and incubated at 37.0 C for 2 h. Finally,
tubes were centrifuged, and resulting supernatants were diluted in 2 ml
culture medium (to achieve estimated concentrations of 0.5% e-16 serum
by volume or 1 x 10-10 M
corticosterone). These 2-ml treatments were placed on day 12 pituitary
cells for 48 h to test bioactivity.
Statistical analysis
Data were analyzed by ANOVA using the general linear models
procedure of SAS (11). The main effects tested were trial and
treatment. Differences between treatments were then tested by
orthogonal contrasts. Differences were considered significant at
P < 0.05. All data presented are the least squares
means and SEs from the ANOVA of at least three independent
replicate experiments.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Unfractionated serum from e-16 embryos
significantly stimulated somatotroph differentiation, with 7.8 ±
0.9% of the population secreting GH (mean ± SE;
n = 3). The bioactivity eluted as a single peak with a relative
molecular mass less than that of the smallest standard (bacitracin;
1.45 kDa), and with maximal induction of somatotroph differentiation of
11.1 ± 0.9% of all pituitary cells. Next, we tested whether the
bioactivity was sensitive to proteolytic digestion. The results are
presented in Fig. 2. Untreated serum from
e-16 embryos, but not from e-12 embryos, significantly stimulated
somatotroph differentiation (Fig. 2A). Trypsin digestion increased the
effect of e-16 serum and exposed somatotroph-differentiating activity
in serum from e-12 embryos. The effect of trypsin digestion on GH
accumulation in culture medium is shown in Fig. 2B. Untreated serum
from e-16, but not e-12, embryos stimulated GH secretion (10.1 ±
0.3 ng/ml with e-16 compared with 5.6 ± 0.3 ng/ml with e-12 serum
and 3.6 ± 0.3 ng/ml under basal conditions). Digestion with
trypsin increased the effectiveness of both e-12 and e-16 serum on
medium GH content. However, e-16 serum remained significantly more
effective than e-12 serum.
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. Heating to 70.0 C for 1 h
increased the bioactivity of e-16 serum and activated GH
cell-differentiating activity in e-12 serum. Moreover, all of the
bioactivity in serum was contained in the ether phase, regardless of
age, whereas none was detected in the aqueous phase of serum.
Bioactivity in the ether-soluble fraction from e-16 serum was greater
than that in the ether phase of e-12 serum (P < 0.05).
In contrast, the effect of ether extraction on bioactivity in e-12
serum (a 4.4-fold increase in activity) was far more dramatic than the
effect of ether extraction on the activity of day 16 serum (a 75%
increase in activity). Heating and ether extraction also increased the
stimulatory effect of the serum on GH accumulation in culture medium
(Fig. 3B). Heated e-12 serum and ether-extracted e-12 serum increased
GH accumulation more than untreated e-12 serum and the aqueous phase of
e-12 serum. Similarly, heating and ether extraction increased activity
in e-16 serum. All of the activity was again found in the ether phase,
which increased GH accumulation to 18.4 ± 0.4 ng/ml compared with
3.7 ± 0.4 ng/ml in cultures treated with the e-16 aqueous phase.
Interestingly, the ether phase of e-16 serum remained more effective in
stimulating GH accumulation than the ether phase of e-12 serum. Taken
together, we conclude that the somatotroph-differentiating activity in
serum is an ether-soluble compound, bound or inhibited by a
trypsin-sensitive protein. Furthermore, the results indicated that
bioactivity in the ether extract was greater on day 16 than on day 12,
whereas levels of the binding/inhibiting protein were greater on day 12
than on day 16. This biochemical profile is indicative of steroid
hormones, which are typically bound to serum proteins.
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). The maximal
responses to glucocorticoids and progesterone were greater than that to
estradiol. Testosterone had a marginal effect at 10-9
M, which was not evident with its stable analog, DHT.
Aldosterone was also found to have no effect (data not shown).
Glucocorticoids and progesterone stimulated GH accumulation in culture
medium to about 7.5 ng/ml compared with the basal level (2.4 ng/ml),
whereas estradiol, DHT, and testosterone failed to do so (data not
shown).
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Although tamoxifen was able to abolish the stimulatory effect of
estradiol on somatotroph differentiation, two doses of the antagonist
failed to block the response to e-16 serum. Next, we tested the effect
of the steroid receptor antagonist, RU486 (Fig. 6). RU486 blocked the effects of
corticosterone and progesterone on somatotroph differentiation. More
importantly, RU486 (10-6 M) abolished the
stimulatory effect of e-16 serum. GH accumulation in the culture medium
in response to e-16 serum was also abolished in the presence of RU486
(17.4 ± 1.7 ng/ml with serum alone and 5.1 ± 1.7 ng/ml with
serum and RU486, compared with 4.6 ± 1.7 ng/ml under basal
conditions). These results indicate that the active compounds in e-16
serum, corticosterone and progesterone, stimulate somatotroph
differentiation through the glucocorticoid receptor.
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. The stimulatory
effects of heated e-16 serum on somatotroph percentage were suppressed
after incubation with corticosterone antiserum. The GH antiserum
control had no effect on serum-induced GH cell differentiation. These
results indicate that corticosterone is the active compound in
embryonic serum that stimulates somatotroph differentiation.
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). The lowest effective dose was 2
x 10-11 M, and the most effective dose to
stimulate GH cell differentiation was 2.5 x 10-9
M.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We found that the somatotroph-differentiating activity in chicken embryonic serum was a small, ether-soluble compound, bound or inhibited by a trypsin-sensitive protein. This biochemical profile is typical of a steroid hormone. Previous reports using rats have indicated that glucocorticoids can induce premature expression of GH protein in vitro (2, 3) and messenger RNA in vivo (4) and in vitro (5). In the present study, we showed with anterior pituitary cells from chicken embryos that somatotroph differentiation can be stimulated by several steroids, including corticosterone, 17ß-estradiol, and progesterone. The effect of estradiol was successfully blocked with the estradiol receptor antagonist tamoxifen. However, tamoxifen did not affect the response to day 16 serum, indicating that the blood-borne, somatotroph-differentiating signal did not function through the estrogen receptor. The role of the glucocorticoid receptor in the serum response was evaluated using RU486. Progesterone and glucocorticoids have been reported to activate each other’s receptors (14), and the receptor antagonist RU486 blocks both receptors in mammals. However, RU486 interacts only with the glucocorticoid receptor in chickens (14, 15). As RU486 blocked both corticosterone and the progesterone induction of somatotroph differentiation in the present study, both compounds were shown to act via the glucocorticoid receptor. More importantly, RU486 abolished the stimulatory effect of day 16 serum, indicating that the active steroid in embryonic serum functioned through the glucocorticoid receptor. After demonstrating this requirement for the glucocorticoid receptor, we tested whether the blood-borne compound was corticosterone. Incubation of day 16 serum and corticosterone with antiserum against corticosterone suppressed the effect of corticosterone and, more importantly, the stimulatory effect of day 16 serum. In contrast, the GH antiserum, used as a control for serum effects, had no effect on the embryonic serum response. Therefore, the blood-borne, somatotroph-differentiating compound in chicken embryonic serum is corticosterone.
The present study along with our previous results (7) indicate that levels of somatotroph-differentiating activity in the serum increase between days 12 and 16 of embryonic development, the period of normal somatotroph differentiation (6). This conclusion is supported by bioactivity detected in untreated serum and serum after heating, ether extraction, or trypsin digestion. Interestingly, levels of adrenal steroids increase around day 15 of chicken embryonic development (16, 17) and day 17 of rat embryonic development (18), just before somatotroph differentiation in both species. This correlation between an increase in serum concentrations of corticosterone, shown to be the blood-borne somatotroph-differentiating compound in the present study, and the ontogeny of GH-secreting cells during normal development suggests that increased adrenal glucocorticoid secretion stimulates the differentiation of pituitary somatotrophs. However, we observed in the present study that levels of a corticosterone-binding/inhibiting protein decreased between embryonic days 12 and 16. Thus, somatotroph differentiation during normal embryonic development could occur in response to an increase in adrenal corticosterone production, a decrease in serum corticosterone-binding/inhibiting activity, or a combination of both events. Additional research is necessary to distinguish between these possibilities.
The mechanism underlying glucocorticoid induction of somatotroph differentiation has not been defined. The effects of glucocorticoids on GH gene expression vary between in vitro and in vivo preparations. In most vertebrate species studied, glucocorticoids stimulate GH gene expression in intact animals (17, 19). In contrast, the effects of glucocorticoids on GH gene expression seem to be culture and cell line dependent (20). The lack of stimulatory effects of glucocorticoids on GH gene expression in culture might be due to the absence of synergism with thyroid hormones. Treatment with DEX in the absence of thyroid hormone stimulated GH expression less than simultaneous DEX and thyroid hormone treatment. However, premature expression of the GH gene cannot be stimulated by thyroid hormones without the presence of endogenous or exogenous glucocorticoids (4). In the present study, we demonstrated that glucocorticoids can induce GH cell differentiation in the absence of thyroid hormones. In our previous report (7), we noted that day 16 embryonic serum induced GH cell differentiation, as detected by immunocytochemistry and RHPAs without GHRH. Therefore, the newly recruited somatotrophs, induced to differentiate by corticosterone, synthesize and release GH in the absence of GHRH. This was also shown in the present study by RIA, in that day 16 serum and corticosterone increased medium GH content. Thus, three different assays (RHPA, RIA, and immunocytochemistry) indicate that somatotroph differentiation occurred in response to corticosterone and in the absence of GHRH. Our laboratory is currently exploring the possibility that corticosterone can interact with other factors to alter the extent of somatotroph differentiation.
Therefore, we conclude that somatotroph differentiation in the chicken embryonic pituitary can by induced in vitro by glucocorticoids and that corticosterone is the active compound responsible for the observed GH cell-differentiating activity of day 16 embryonic serum. The fact that normal GH cell differentiation during embryonic development occurs after an increase in circulating corticosterone levels in chickens (17) and rats (18) supports the involvement of glucocorticoids in GH cell differentiation during normal development in vivo.
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Footnotes |
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Received June 5, 1997.
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