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The Perinatal Research Laboratory (F.M.P., J.S., J.C.R.), Department of Physiology and Pharmacology (F.M.P., J.S., J.C.R.), and Department of Obstetrics and Gynecology (J.S., J.C.R.), The Bowman Gray School of Medicine, Winston-Salem, North Carolina 27157
Address all correspondence and requests for reprints to: Dr. Frank M. Perez, Assistant Professor, Department of Physiology and Pharmacology, The Bowman Gray School of Medicine, Medical Center Boulevard, Winston-Salem, North Carolina 27157. E-mail: fperez{at}bgsm.edu
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Abstract |
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Cell blot analysis of individual corticotrophs identified two types of secretory responses (increases in the number of secreting cells and average amount of ACTH released per cell) to CRH or AVP that changed during fetal development. At 100 d of gestation, CRH increased the proportion of secreting cells from 65 ± 3% (no test agent) to approximately 90%; AVP exerted a negligible effect on the relative abundance of secreting cells. At 120 d of gestation, both secretagogues, alone or in combination, increased the proportion of secreting corticotrophs from 49 ± 6% to about 85%. At 135 d of gestation and in the adult, AVP, alone or in combination with CRH, increased secreting corticotrophs from about 53 ± 6% to about 80%. CRH alone exerted a nominal effect on the proportion of secreting cells. Additional analyses showed that, at 100 or 120 d of gestation, the average amount of ACTH secreted by individual corticotrophs did not change in response to CRH or AVP. However, near term and into adulthood, the average quantity of ACTH released from individual cells increased in response to these agents.
Our findings suggest that maturational changes in fetal corticotrophs dictate whether their secretory response to CRH or AVP results from an increase in the proportion of cells secreting ACTH and (or) an increase in the average amount of hormone secreted by individual cells.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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These developmental changes in corticotroph morphology are accompanied by alterations in their response to hypothalamic regulatory factors. In this regard, we recently reported (4) that corticotrophs at 108 d of gestation are relatively more responsive to CRH than to AVP, whereas those at 135 d and in the adult are more responsive to AVP.
These findings were obtained by measuring cumulative responses of cell populations to CRH and AVP in vitro. To further investigate corticotrophs during development, we used an immunoblotting technique to study individual cells. Other investigators (5, 6, 7, 8, 9) have used this method to characterize secretion of hormones and small peptides by single pituitary cells. We used cell immunoblotting in the present study to determine whether corticotrophs are heterogeneous in terms of basal ACTH secretion and response to CRH and AVP.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Preparation of pituitary cells
Individual cells were prepared from adult and fetal anterior
pituitaries according to a method described elsewhere (4). Minced
tissue fragments were placed in HEPES-buffer containing 0.4%
collagenase (Worthington Biochemical Corp., Freehold, NJ) and DNAase I
(Sigma Chemicals, St. Louis, MO) and incubated for 2h at 37 C with
gyratory shaking. After incubation, the fragments were passed gently
through a flame-tapered glass pipet and the dispersed cells were washed
in DMEM/Ham’s F12 containing 0.2% polypep. Cells were filtered to
eliminate clumps and reaggregates and treated as described below.
Cell culture
Adult and fetal sheep pituitary cells were divided into three
groups: those cultured on glass cover slips, coated plastic surfaces,
or a protein-capturing membrane (Immobilon, Millipore, Bedford, MA).
Cells of the first group were used to measure the proportion of
corticotrophs in the total cell population. Cells were cultured
according to a modified method of Wilfinger (10). This procedure
consisted of incubating 3.75 x 104 cells in a 50-µl
droplet of medium on a glass cover slip (9 x 9 mm2;
Bellco Glass, Inc., Vineland, NJ) at the bottom of a well (24-well
tissue culture plate; Corning, Corning, NY). After 1 h for the
cells to attach to the glass, the well was filled with medium (1.0 ml)
and the cells were incubated for an additional 1 h. After this
step, the cells were fixed for 1 h, as described below, and
processed by immunocytochemistry for ACTH.
Cells of the second group were used to measure cumulative responses to hypothalamic stimulatory factors and cultured at a high cell density (1.5 x 105 cells/28 mm2) on poly-L-lysine-coated plastic surfaces (24-well tissue culture plate) according to the method of Perez (11). Cells were incubated for 2 h, washed once, and then treated with fresh medium containing vehicle (control), CRH (10 nM), AVP (100 nM), or both agents together. After 2 h, the medium was collected and centrifuged, and the supernatant solutions were stored at -20 C for ACTH assay.
Cells of the third group were used to measure the response of individual corticotrophs to hypothalamic stimulatory factors. Cells were cultured on Immobilon; a droplet (100 µl) of medium containing cells (1.5 x 104) was applied to the membrane. After 15 min, CRH (10 nM, final concentration), AVP (100 nM), or both agents together (10 µl each) were added directly into the droplet. Medium alone (10 µl) was added to the control droplets. After 2 h, the cells, attached to the membrane, were fixed (1 h) with paraformaldehyde (4.0%) and processed by immunocytochemistry for ACTH.
ACTH immunocytochemistry
Pituitary cells, attached to cover slips, were stained for ACTH
according to a modified method of Denef et al. (12). Cells
were fixed for 1 h with Bouin’s solution (1.0 ml), washed, and
incubated overnight in TRIS/NaCl buffer containing primary antiserum
(rabbit antisheep ACTH; 1:3,000). After this step, they were washed and
incubated for 2 h with secondary antiserum (goat antirabbit
IgG/HRP; 1:500). Immunoreaction product was developed with DAB and
0.006% H2O2; cells were counterstained with
hematoxylin.
Pituitary cells, attached to Immobilon, were immunostained for ACTH according to a modified method of Denef et al. (12). Cells were fixed with glutaraldehyde (2.5%), incubated with primary (1:1500) and secondary (1:500) antisera, reacted with DAB/H2O2, and counterstained with hematoxylin.
Controls for all immunocytochemical staining procedures included: 1) replacement of the primary antiserum with nonimmune rabbit serum; 2) preadsorption of the primary antiserum with purified ACTH before its use; and 3) immunolocalization of purified ACTH on Immobilon. The ACTH antiserum that was used in these studies was prepared and described elsewhere (13).
ACTH cell analysis
A minimum of 500 cells on cover slips or Immobilon membranes was
included for analysis of each treatment by using a 40 x objective
and bright-field illumination. A serpentine surveying pattern was used
to insure that each cell was counted only once. Cells on cover slips
and Immobilon were characterized as either ACTH immunopositive or
immunonegative.
After determining that analysis of cells on cover slips or Immobilon
yielded similar results, we used the former condition to measure the
proportion of corticotrophs in the total cell population. Cells on
Immobilon were used to determine the proportion of corticotrophs that
secreted ACTH onto the membrane. Cells that were associated with ACTH
on the membrane were counted as secretors. ACTH release by individual
cells (see Fig. 7
) was calculated by dividing the amount of hormone
secreted by cell populations (measured by RIA) by the number of
secretors (determined by immunoblotting).
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0.05 level of significance. Synergism was tested
by statistically comparing the net response of cells with CRH and AVP
together with the arithmetic sum of the net responses to each peptide
alone. |
Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
Corticotroph frequency was 22 ± 1%, 18 ± 1%, and 14
± 1% at 100, 120, and 135 d of gestation, respectively. The
frequency (9 ± 0%) of corticotrophs in the adult did not differ
from that of late gestation.
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shows a representative immunoblot of
enzymatically-dispersed sheep pituitary cells. Cells that contained and
released ACTH were readily identifiable on the basis of ACTH
immunoreactivity. Individual corticotrophs stained intensely dark-brown
and the amount of ACTH that was secreted on the membrane varied (as
determined by computerized densitometry; data not shown).
Immunonegative cells were stained only light-blue by hematoxylin. All
cells that stained positively for secreted ACTH also were
immunopositive for content. Immunoreactivity was not observed when the
primary antiserum was replaced with nonimmune serum or when the primary
antiserum was preadsorbed with ACTH (data not shown).
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, upper panel). These agents together
increased ACTH release by 144 ± 6% above the control value.
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, lower
panel). This percentage was increased to 92 ± 4% by CRH; in
contrast, the proportion of corticotrophs responding to AVP (76 ±
5%) was not different from the basal value. The combined effect of
these agents on the proportion of secreting corticotrophs was similar
to that of CRH alone.
In cells from 120 d of gestation, CRH or AVP by themselves each
increased ACTH release in cell populations by about 80% above the
control value (Fig. 4, upper panel). The
combined effect of these agents on ACTH release was similar to either
one alone. Studies of individual corticotrophs showed similar responses
to CRH and AVP (lower panel). Both agents, alone or in
combination, increased the proportion of secreting corticotrophs from
49 ± 6% (control value) to about 85% of the total ACTH cell
population.
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, upper panel). The response of cells to
these agents in combination (226 ± 43%) resembled that of AVP
alone. Single-cell studies showed a parallel secretory profile
(lower panel): 53% ± 6% of all corticotrophs secreted
ACTH under basal conditions. CRH had no effect on the percentage of
secreting corticotrophs; AVP increased it to 82 ± 4% These
agents in combination increased the percentage of secreting cells to a
value (77 ± 6%) that was similar to that of AVP alone.
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, upper panel). The combined effect of
these agents on ACTH release was 554 ± 9%. Single-cell studies
showed that 56 ± 4% of all corticotrophs secreted ACTH under
basal conditions (lower panel). CRH alone had no effect on
the proportion of secreting cells; AVP increased it to 82 ± 4%.
There was no difference between the response of the cells to these
agents in combination and that to AVP alone.
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shows the calculated average of ACTH release by
individual corticotrophs as determined by expressing hormone secretion
by cell populations per individual secreting corticotroph. This
analysis showed that, at 100 and 120 d of gestation, CRH and (or)
AVP did not alter ACTH secretion per secreting corticotroph. However,
at 135 d of gestation, AVP alone increased ACTH release per
secreting corticotroph by 240 ± 80% over the control value and
by 160 ± 40% in combination with CRH. In the adult, CRH or AVP
increased ACTH release by 121 ± 10% and 107 ± 10%,
respectively; these agents were additive (292 ± 10%)
together. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, lower
panels) or increases in the amount of ACTH released by individual
cells (Fig. 7), change during fetal development and in adulthood. Thus,
maturation of the response of corticotrophs to CRH or AVP depends, in
part, on changes among the population of ACTH-secreting cells that
occur as a function of gestational age. This finding is consistent with
other in vitro ontological studies (4) reporting that fetal
sheep corticotrophs at 108 ± 5 d of gestation respond to CRH
or AVP, but by 139 d, they respond significantly only to AVP.
Both types of secretory responses of sheep corticotrophs to CRH
(i.e. increases in numbers of secreting cells or secretory
output per cell) have been reported also for adult rat pituitary cells.
Leong and colleagues (14, 15, 16) found that CRH alters both the number of
ACTH-secreting cells and the amount of ACTH secreted by a fixed number
of corticotrophs. Childs and Burke (17) reported that CRH increases the
percentage of secreting corticotrophs and speculated that the
secretagogue recruits a subpopulation of quiescent cells to release
ACTH. Our immunoblotting studies support this idea by identifying a
subset of corticotrophs that had no associated secretory product on the
membrane. Although it is possible that these quiescent cells released
ACTH in amounts that fall below the detection limit of the cell blot
assay, our results suggest that CRH stimulates a subpopulation of
corticotrophs to release ACTH. This type of secretory response occurs
at 100 and 120 d of gestation (Figs. 3 and 4). As the fetus
matures, the secretory response changes to one that involves increased
output of ACTH from already-secreting cells (Fig. 7).
Developmental responses of sheep corticotrophs to AVP involved changes
in both the proportion of secreting cells (
Figs. 4–6) and amount of
ACTH released per cell (Fig. 7). The former secretory response has not
been observed in rat pituitary cells and might reflect species
differences in the regulation of ACTH secretion (16). Increases in ACTH
output by individual cells responding to AVP have been reported for rat
corticotrophs (16).
Developmental changes in secretory responses of corticotrophs to CRH
and AVP reported herein coincide with increases in the total number of
anterior pituitary cells, decreases in cell frequency (as shown in Fig. 1 and reported by others), and alterations in cell size (1, 2, 3). Whether
these morphological changes correspond to differences in functional
subtypes of corticotrophs remains unknown. However, our findings raise
the possibility that maturation of the HPA axis involves alterations in
subpopulations of secreting corticotrophs and amount of ACTH released
by individual cells.
Several mechanisms might be involved in changing the response of ovine
corticotrophs to CRH and AVP during development. For example, the
increased responsiveness of late gestational fetal and adult
corticotrophs to AVP complements radioreceptor bindings studies of Shen
et al. (18). These investigators reported that twice as many
AVP receptors are present on sheep, compared with rat, anterior
pituitary membranes, with both receptors showing similar affinities
for the hormone. Similarly, diminishing responsiveness of late
gestational corticotrophs to CRH (Fig. 5) is consistent with decreases
in the number of CRH receptors near term (19).
These findings raise the possibility that developmental changes in corticotroph responses, in part, are caused by expression of CRH and AVP receptors. The mechanism(s) controlling expression of these receptors during development of the fetal sheep is unclear but may involve cortisol and other steroids (20, 21, 22, 23). Additional mechanisms that might change corticotroph responses to CRH or AVP include alterations in signal transduction, cell-to-cell interactions, and differential release of these and other trophic factors by the hypothalamus (15, 24, 25, 26).
Concentrations of CRH and AVP used in these studies were selected on the basis of in vitro experiments with adult sheep anterior pituitary cells and in vivo measurements of concentrations in portal blood. We used concentrations that would produce measurable responses and be within a physiological range. In adult sheep, CRH (10 nM) and AVP (100 nM) induce the maximum secretory response from corticotrophs in vitro (27). In portal blood, AVP is usually found in excess to CRH at rest and in response to stress (28). We used the same concentrations of CRH and AVP, at all ages studied, to enable comparisons across ages. It remains possible that higher concentrations of CRH could provoke responses from corticotrophs at ages where none were observed at the concentration used in this study. It is also possible that corticotrophs of pregnant ewes may respond to CRH and AVP differently from those of adult nonpregnant female or male sheep, but experience to date with tests of in vitro ACTH secretion suggests that this is unlikely.
In summary, we have identified at least two modes by which sheep corticotrophs responded to CRH and AVP: increases in the number of secreting cells and quantity of ACTH released by single cells. The type of secretory response changed during fetal development and in adulthood. Our findings suggest that maturation of the HPA axis involves alterations in responses of corticotrophs to CRH and AVP.
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Acknowledgments |
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Footnotes |
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Received September 9, 1996.
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References |
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-MSH, -MSH, ACTH and
ß-endorphin/ß-lipotrophin in the fetal sheep pituitary: an
ontogenic study. J Dev Physiol 8:355–368[Medline]
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