Divergent changes in plasma ACTH and pituitary POMC mRNA after
cortisol administration to late-gestation ovine fetus
T. M.
Jeffray1,2,3,
S. G.
Matthews1,
G. L.
Hammond2,4, and
J. R. G.
Challis1,2,3
1 Departments of Physiology and
Obstetrics and Gynaecology, University of Toronto, Toronto M2S
1A8; 2 Medical Research
Council Group in Fetal and Neonatal Health and Development,
3 Lawson Research Institute and
Departments of Obstetrics and Gynaecology, and Physiology, and
4 London Regional Cancer Centre,
Victoria Hospital, Departments of Obstetrics and Gynaecology,
Biochemistry, and Oncology, University of Western Ontario, London
N6A 5C1, Ontario, Canada
 |
ABSTRACT |
Plasma concentrations of cortisol and
adrenocorticotropic hormone (ACTH) rise in the late-gestation sheep
fetus at approximately the same time as there is an increase in the
plasma levels of corticosteroid- binding globulin (CBG). We
hypothesized that intrafetal cortisol infusion during late pregnancy
would stimulate an increase in fetal plasma CBG, which in turn would
bind cortisol and diminish glucocorticoid negative-feedback regulation
of the fetal pituitary, leading to an increase in plasma ACTH
concentrations. Cortisol was infused into chronically catheterized
fetal sheep beginning at 126.1 ± 0.5 days of gestation and
continued for 96 h. Control fetuses were infused with saline. In
cortisol-infused fetuses, the plasma cortisol concentrations rose
significantly from control levels (4.4 ± 0.6 ng/ml) to 19.3 ± 3.1 ng/ml within 24 h and remained significantly elevated throughout
the infusion period. Plasma immunoreactive (ir) ACTH concentrations
were significantly elevated in cortisol-infused fetuses within
24-48 h and remained significantly higher than in controls
throughout the 96-h experimental period. Plasma free cortisol
concentrations increased 10-fold and remained significantly elevated in
cortisol-infused animals, despite a rise in plasma
corticosteroid-binding capacity. Levels of pituitary proopiomelanocortin (POMC) mRNA in the fetal pars distalis and pars
intermedia were 96 and 38% lower, respectively, after 96 h of cortisol
infusion. Therefore physiological elevations of plasma cortisol, in the
late-gestation ovine fetus, lead to increases in mean plasma irACTH
concentrations, but this is not associated with increases in fetal
pituitary POMC mRNA levels.
corticosteroid-binding globulin; free cortisol; adrenocorticotropic
hormone; proopiomelanocortin
 |
INTRODUCTION |
IN FETAL SHEEP, basal plasma concentrations of
immunoreactive (ir) adrenocorticotropic hormone (ACTH) and cortisol
(32) and pituitary proopiomelanocortin (POMC) expression (29) rise concomitantly in late gestation. This occurs despite demonstrable negative-feedback effects of glucocorticoids on stress-induced (1) or
corticotropin-releasing hormone (CRH)-stimulated pituitary-adrenal function (31), suggesting that there may be a decrease in cortisol negative feedback on the hypothalamus and pituitary at this time. Apostolakis et al. (3) previously showed that administration of
cortisol to the ovine fetus at 134 days of gestation altered ACTH
pulsatility, increasing ACTH pulse peak and nadir values (3). However,
the mechanisms whereby intrafetal cortisol has a positive
effect on plasma ACTH concentrations during late gestation is not
clear. Therefore we infused cortisol to fetal sheep, beginning before
the prepartum rise in endogenous cortisol, in amounts that would
reproduce plasma cortisol concentrations similar to those near term to
determine the effects on plasma ACTH and to examine the underlying
mechanisms of any changes.
The plasma concentration of corticosteroid-binding globulin (CBG), the
high-affinity binding protein for cortisol, also rises during the last
third of gestation (4, 7), reflecting an increase in its synthesis in
the fetal liver (7). We have previously showed that CBG biosynthesis
was stimulated by exogenous cortisol (5, 23) and reduced after
bilateral adrenalectomy of the ovine fetus (23). It was therefore
suggested that the rise in cortisol stimulated CBG and would in turn
help to maintain a low free cortisol concentration in plasma despite
elevations in the total (free + bound) cortisol concentration (4, 11).
We reasoned that a rise in CBG would result in decreased free cortisol
(11, 26), which in turn would diminish the negative-feedback effects of
cortisol on the pituitary, resulting in an increase in pituitary POMC
mRNA levels and ACTH output. To examine the underlying mechanisms of
the ACTH response to exogenous cortisol, we measured levels of POMC
mRNA in different regions of the pituitary and CBG mRNA in the liver at
various times during an intrafetal cortisol infusion.
Therefore the overall hypothesis of the present study was that
intrafetal cortisol administration during late gestation would increase
hepatic CBG synthesis and circulating CBG levels, thereby reducing free
cortisol concentrations in plasma. In turn, this would reduce the
negative-feedback effects of cortisol on pituitary POMC expression and
irACTH output, resulting in an increase in circulating irACTH
concentrations, despite elevated total cortisol concentrations in
plasma. In this manner, we would approximate the plasma profiles of
ACTH and cortisol observed in the fetal sheep near term.
 |
METHODS |
Animals.
Surgery was performed under general anesthesia, on mixed breed ewes, at
days 119-122 of gestation (full
term is 145-147 days). The techniques used have been described
previously (14). Briefly, a midline incision was made in the ewe's
lower abdomen to expose the uterus, which was then opened, and the
fetal head was exteriorized. Polyvinyl catheters, filled with sterile
heparinized saline, were inserted into a fetal carotid artery and
jugular vein. Catheters were also placed into the amniotic cavity and a
maternal femoral vein. Uterine electromyographic (EMG) leads (Cooner
Wire, Chatsworth, CA) were attached to the uterus to monitor myometrial
electrical activity. Prophylactic antibiotics were administered at the
time of surgery and continued for 3 days postoperatively as described previously (14).
Blood samples were collected daily, and pH,
PCO2, and
PO2 were measured using an ABL-5
blood gas analyzer (Radiometer, Copenhagen, Denmark). The protocols
were approved by the Animal Care Committees of St. Joseph's Health
Centre, University of Western Ontario, and University of Toronto in
accordance with the guidelines of the Canadian Council on Animal Care.
Experimental protocols.
Animals were allowed a minimum of 5 days to recover after surgery
before experimentation began. Starting on days
124-129, fetuses received an intravenous infusion
of either cortisol (11
,17,21-trihydroxy-4-pregnene-3,20-dione, Steraloids, Wilton, NH; 5 µg/min, n = 7) or an equal volume of saline (3 ml/h, with 2% vol/vol ethanol,
n = 6) for 96 h. Fetal arterial blood
samples (4 ml) were collected into chilled, heparinized syringes every
8 h beginning 24 h before the start of infusion and continuing
throughout the experiment. Samples were centrifuged immediately at
1,500 g for 10 min at 4°C, and the
plasma was stored at
20°C until analysis. Additional animals
were infused for 12 (n = 4) or 24 h
(n = 4) to examine changes in POMC and
CBG mRNA levels associated with the initial phase of the rise in plasma cortisol concentrations. At the conclusion of the infusion periods (12, 24, or 96 h), the animals were killed with an overdose of 24%
pentobarbital sodium (Euthanyl, MTC Pharmaceuticals, Cambridge, ON,
Canada), and fetal tissues were collected quickly. Fetal pituitaries were frozen on dry ice for in situ hybridization and
immunohistochemistry. A portion of the right lobe of the liver was
frozen rapidly in liquid nitrogen for Northern blot analysis.
Fetal blood gases and uterine activity.
Fetal blood pressure and amniotic fluid pressure were measured
continuously using Statham pressure transducers (P23XL, Spectramed, Oxnard, CA) and displayed on a chart recorder (model 78D, Grass Instrument). Cortisol has previously been shown to induce hypertension in the ovine fetus (17). Therefore blood pressure was monitored to
determine whether there was any association between mean arterial pressure and changes in plasma hormone values. Mean fetal blood pressure was calculated as 0.4 × (systolic pressure
diastolic pressure) + diastolic pressure
amniotic fluid
pressure. Blood pressure was measured at five different time points
every hour, and the mean was calculated. The daily mean was then
calculated from these hourly values. Because intrafetal administration
of cortisol can induce premature parturition in sheep (25), uterine activity was monitored continuously to ensure that any changes in
plasma irACTH concentrations were not attributable to the process of
labor. Uterine EMG activity was measured using a Grass wide-band AC
preamplifier (Grass model 78D) and was recorded as the number of
episodes of low-amplitude activity lasting >5 min, referred to as
"contractures" (30), and the number of contractions (activity lasting 0.5-1 min) per 2-h period (21).
Measurement of plasma ACTH, cortisol, free cortisol, and CBG.
Plasma irACTH concentrations were measured by a commercial
radioimmunoassay (RIA) kit (Incstar, Stillwater, MN) that was validated previously for use in the fetal sheep (32). The intra- and interassay coefficients of variation were 9 and 13%, respectively, and
the mean assay sensitivity was 6.5 pg/ml. This ACTH antibody
cross-reacts <0.01% with
-melanocyte-stimulating hormone (MSH),
-MSH,
-endorphin, and
-lipotropin (
-LPH; Incstar) and does
not recognize pro-ACTH or POMC (kindly provided by Dr. J. Schwartz,
Bowman Gray School of Medicine, Winston-Salem, NC). The antibody
recognized >95% immunoreactivity corresponding to ACTH-(1
39) when
samples of fetal sheep plasma in normoxemia or during hypoxemia were
assayed after high-performance liquid chromatography separation of
ACTH-related peptides (12). Plasma cortisol concentrations were
quantified by RIA after extraction with diethyl ether. The antibody
characteristics and assay validation for measurement of cortisol in
fetal sheep plasma have been described previously (14). The combined
intra- and interassay coefficient of variation was 12%. The percentage of free cortisol in duplicate samples of fetal plasma was measured by
the centrifugal ultrafiltration-dialysis technique developed and
described by Hammond et al. (20). All of the samples were measured in a
single assay. Fetal plasma CBG levels were measured as corticosteroid
binding capacity (CBC) determined using the saturation binding assay of
Ballard et al. (4), with modifications described previously (13). The
within- assay coefficient of variation was <10%.
Northern blot analysis of CBG mRNA.
We previously showed that the liver is the major site of CBG
biosynthesis in fetal sheep (7). Total cellular RNA was extracted from
the fetal liver, electrophoresed, and blotted as described previously
(6). The blotting membranes were probed using an ovine CBG cDNA (6).
After autoradiographic exposure, the blots were stripped and reprobed
with a cDNA to mouse 18S rRNA to allow correction for variations in gel
loading and transfer. The relative optical densities (ROD) were
determined using computerized image analysis (Imaging Research, St.
Catharines, ON, Canada). Results are expressed as the ratio of the ROD
of the CBG mRNA to 18S rRNA hybridization signals.
In situ hybridization of pituitary POMC mRNA.
Frozen pituitaries were sectioned (15-µm coronal sections) using a
cryostat (Tissue-Tek, Miles Canada, Etobicoke, ON, Canada), mounted
onto poly-L-lysine (Sigma
Chemical, St. Louis, MO)-coated slides and fixed in 4%
paraformaldehyde (28). Pituitary sections were incubated with a
35S-labeled, 45-mer
oligonucleotide complementary to bases 711-756 of the porcine POMC
gene (19) as previously described in detail (37). The slides were
exposed to autoradiographic film (XAR 5, Kodak) for 2 h at room
temperature to determine the hybridization signal for POMC mRNA levels
in the pars intermedia and then reexposed for 4 days to measure the
levels of POMC mRNA in the pars distalis. The two exposure times were
necessary for the hybridization signal to be within the linear range of
the film for the two regions of the pituitary. Linearity was
established by the simultaneous exposure of the film to
14C standards (27). The
autoradiograms were then analyzed using computerized image analysis
(Imaging Research). Results are expressed as ROD for a minimum of 15 pituitary sections per animal. A control 45-mer sense oligonucleotide
was also synthesized, and no signal was observed when it was hybridized
with pituitary sections (10). The fetal pituitary sections were then
coated with Ilford K5 liquid emulsion, developed and counterstained
with Carazzi's hematoxylin to identify nuclei. The silver grain
deposits were visualized using light microscopy.
Immunohistochemistry.
Immunohistochemical detection of irACTH was performed on 15-µm frozen
pituitary sections prepared as described above for in situ
hybridization. A polyclonal antibody to human ACTH-(1
24) (Dako,
Carpinteria, CA) was used in conjunction with avidin-biotin-peroxidase reagents from the Vectastain ABC kit (Vector Laboratories, Burlingame, CA), as previously described (33). The ACTH antibody has been characterized extensively, and the antigenic site has been shown to be
between amino acids 18 and 24 (22). This ACTH antibody cross-reacts
<1% with
-MSH,
-MSH, and
-LPH (22). The number of
immunopositive cells within 10 fields (475 µm × 350 µm = 1.7 × 105
µm2) was counted for each
animal. Adjacent sections were incubated with the primary antibody in
the presence of an excess of antigen [human ACTH-(1
24)]
to provide negative controls.
Data analysis.
Blood pressure and plasma hormone concentrations were measured in
samples collected every 8 h, and the daily mean value was calculated
for each animal. These values are reported as means ± SE for the
number of animals stated. The maximal and minimal changes in irACTH
concentrations were calculated from the three plasma samples collected
each day, relative to the mean plasma irACTH value during the control
period, for individual animals and reported as means ± SE. Changes
in plasma cortisol, CBG, irACTH, maximal and minimal change in irACTH,
mean arterial pressure, and uterine activity were analyzed by
two-way analysis of variance corrected for repeated measures.
Statistical significance was determined as
P
0.05. The effects of individual
times of treatment were assessed by Student-Newman-Keuls multiple range
tests. Values for hepatic CBG mRNA levels, free cortisol
concentrations, and number of corticotrophs staining positively for
irACTH were not distributed normally and were therefore assessed by the
Kruskal-Wallis analysis of variance followed by Dunn's test or the
Mann-Whitney rank- sum test (Sigmastat, Jandel Scientific, CA).
 |
RESULTS |
Plasma cortisol concentrations.
The daily mean concentration of cortisol in fetal plasma rose during
cortisol infusion from basal values of 4.4 ± 0.6 to 19.3 ± 3.1 ng/ml within 24 h (n = 6, P < 0.05; Fig.
1). The mean plasma cortisol concentration
rose progressively to a maximal concentration of 38.6 ± 2.7 ng/ml,
which is similar to that seen in the ovine fetus near term (32). Plasma
cortisol concentrations in the control animals did not change
significantly throughout the infusion period.

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Fig. 1.
Plasma cortisol concentrations before and during a 96-h cortisol
(filled bars, n = 7) or saline (open
bars, n = 6) infusion, starting at
time 0, to fetal sheep in late
gestation. Values are means ± SE.
* P < 0.05. Cortisol infusion
increased plasma cortisol concentrations significantly within 24 h
[2-way repeated-measures analysis of variance (ANOVA) followed by
Student-Newman-Keuls] to values similar to those for full-term
animals.
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Fetal blood gases and uterine activity.
Fetal arterial PO2,
PCO2,
O2 saturation, and pH were
unchanged throughout the study in both cortisol- and saline-treated
animals (Table 1). Mean arterial pressure
increased significantly within the first 24 h of cortisol
administration from 42.6 ± 1.0 to 48.7 ± 0.7 mmHg and remained
at this level throughout the infusion period. There was no significant
change in mean arterial pressure in the saline-infused fetuses (Table 1). The number of contractures or contractions per 2-h interval did not
change significantly in the saline-treated fetuses. Similarly, during
intrafetal cortisol infusion, uterine activity was not altered during
the first 72 h (at 48-72 h, mean number of contractures = 3.4 ± 0.2 and contractions = 1.0 ± 0.2 per 2-h period). However, there was an increase in uterine contractility during the final 24 h of
intrafetal cortisol infusion (Table 1).
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Table 1.
Fetal arterial blood gases, mean arterial pressure, and uterine
activity (per 2-h period) during first 24 h before and first and last
24 h of infusion if saline or cortisol
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Effects of cortisol on plasma irACTH.
Plasma irACTH concentrations rose significantly in the cortisol-treated
fetuses from values of 25.4 ± 2.7 pg/ml during the control period
(Fig. 2) to a significantly elevated level
of 37.8 ± 3.9 pg/ml at 24-48 h (Fig. 2). Fetal ACTH values
remained elevated throughout the cortisol infusion period
(n = 7, P < 0.05, effect of time
F = 7.9 and cortisol
F = 9.6; Fig. 2). In the
saline-infused animals, plasma ACTH concentrations did not change
significantly from control values of 27.0 ± 1.7 pg/ml
(n = 6; Fig. 2).

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Fig. 2.
Plasma immunoreactive (ir) ACTH concentrations before and during a 96-h
infusion of cortisol (filled bars, n = 7) or saline (open bars, n = 6),
starting at time 0, to fetal sheep in
late gestation. Values are means ± SE.
* P < 0.05 (for
repeated-measures 2-way ANOVA followed by Student-Newman-Keuls).
Cortisol infusion significantly increased mean irACTH concentrations by
24-48 h, and irACTH remained significantly elevated throughout
infusion compared with saline controls.
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ACTH is secreted in pulses; therefore plasma irACTH concentrations were
variable between samples within individual fetuses. The maximal and
minimal changes in plasma concentrations of irACTH for the three
samples collected each day were calculated relative to the initial 24-h
control period for individual animals. The maximal change in irACTH was
significantly higher at 24-48 h of cortisol infusion and remained
significantly elevated throughout the infusion compared with
saline-treated animals (Table 2). There was
no difference in the minimal change in irACTH for either of the
infusion groups throughout the experiment (data not shown).
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Table 2.
Mean maximal change in plasma irACTH compared with average plasma
irACTH concentrations during 24-h control period
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Effects of cortisol on CBG biosynthesis and secretion.
Plasma CBG levels were similar in both the cortisol (34.6 ± 2.1 ng/ml) and saline-treated animals (27.5 ± 3.0 ng/ml) during the
control period (Fig. 3). The plasma CBG
levels of the cortisol-treated animals were significantly elevated
(51.1 ± 3.2 ng/ml) by 48-72 h of infusion and remained
elevated at 72-96 h (P < 0.05, effect of time F = 6.0 and treatment
F = 46.3).

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Fig. 3.
Plasma corticosteroid-binding capacity (CBC) before and during a 96-h
cortisol (filled bars, n = 7) and
saline (open bars, n = 6) infusion,
starting at time 0, to fetal sheep in
late gestation. Values are means ± SE.
* P < 0.05 (repeated-measures
2-way ANOVA followed by Student-Newman-Keuls).
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Northern blot analysis of RNA from the fetal liver identified a single
CBG transcript of 1.8 kb (Fig.
4A).
Cortisol treatment elevated levels of hepatic CBG mRNA
(P < 0.05, Kruskal-Wallis analysis
of variance). After 96 h, hepatic CBG mRNA levels were significantly
higher (P
0.05, Mann-Whitney
rank-sum test) in the cortisol-infused animals.

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Fig. 4.
Effect of cortisol on hepatic corticosteroid-binding globulin (CBG)
expression. A: Northern blot analysis
of CBG mRNA in livers of individual fetuses treated with cortisol or
saline for 12 (n = 4), 24 (n = 4), or 96 h (saline
n = 6 and cortisol
n = 7). A cDNA probe to 18S rRNA was
used to control for amount of RNA analyzed.
B: histograms show ratio of relative
optical densities (ROD) of CBG mRNA: 18S rRNA for each of cortisol
(filled bars) and saline (open bars) infusion groups. Values are means ± SE. * P < 0.05 (Mann-Whitney U test).
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Plasma free cortisol levels.
The percentage of free cortisol in plasma increased threefold, from 6.3 ± 0.5 to 16.8 ± 6.2% at 8 h of cortisol infusion (Fig. 5A), but
by 72 h the percentage of free cortisol was not significantly different
from that of saline-infused animals. However, the absolute plasma
concentration of free cortisol rose within 8 h and remained significantly elevated in cortisol-treated animals throughout the 96-h
infusion (Fig. 5B). There was no
change in the percentage of free cortisol in the plasma of
saline-treated animals throughout the course of the experiment, and the
absolute free cortisol concentration did not change significantly from
mean values of 0.3 ± 0.1 ng/ml (n = 3; Fig. 5B).

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Fig. 5.
Changes in percentage of free cortisol
(A) and total free cortisol
(B) concentrations in cortisol
(filled bars)- or saline (open bars)-treated animals. Values are means ± SE; n = 6 in each group, except
at 96 h, n = 3 cortisol-infused and
n = 4 control animals.
* P < 0.05.
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Effect of cortisol on levels of pituitary POMC mRNA and irACTH.
Levels of POMC mRNA in the pars distalis of cortisol-treated animals
were lower (Fig.
6C) than
those in saline control animals (Fig.
6A) after 96 h of infusion. POMC
mRNA levels in the pars intermedia were also decreased after 96 h of
cortisol infusion (Fig. 6D) compared
with the saline-treated controls (Fig.
6B).

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Fig. 6.
Autoradiograms of coronal pituitary sections after in situ
hybridization using a 35S-labeled
proopiomelanocortin (POMC) oligonucleotide. Fetuses treated for 96 h
with saline (A and
B) or cortisol
(C and
D).
A and
C were exposed for 4 days to allow
analysis of POMC mRNA in pars distalis.
B and
D were exposed for 2 h to allow
analysis of POMC mRNA in pars intermedia (signal in pars distalis is
barely evident at this time). Scale bar: 470 µm for
A and
C; 222 µm for
B and
D.
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POMC mRNA levels were quantified by computerized image analysis. There
was no significant change in levels of POMC mRNA within the pars
distalis or the pars intermedia after 12 h of cortisol treatment, but
the levels had fallen to 4% of controls by 96 h (Fig.
7). High-resolution analysis using liquid
silver emulsion autoradiography confirmed this finding by the near
absence of silver grain deposits in the pars distalis after 96 h of cortisol infusion (Fig.
8C),
compared with the abundance of silver grain deposits seen in the
saline-treated fetuses (Fig. 8A).
POMC mRNA levels in the pars intermedia of the cortisol-treated animals were unchanged at the end of the first 24 h of infusion, but the levels
had decreased by 38% compared with saline controls after 96 h of
infusion (Fig. 7). There was no apparent change in the number of silver
grains deposited between groups (Fig. 8,
E and G).

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Fig. 7.
Histogram illustrating changes in POMC mRNA levels in pars distalis and
pars intermedia after 96 h of cortisol (filled bars) or saline (open
bars) treatment. Values are expressed as ROD of autoradiograms and
expressed as means ± SE.
* P < 0.05 (Student-Newman-Keuls).
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Fig. 8.
Photographs of emulsion autoradiograms localizing POMC mRNA within pars
distalis (A and
C) and pars intermedia
(E and
G) and irACTH peptide levels in pars
distalis (B and
D) and pars intermedia
(F and
H) of ovine fetus after a 96-h
infusion of cortisol (C,
D, G,
and H) or saline
(A,
B, E,
and F). PI, pars intermedia; PD,
pars distalis. Scale bar: 50 µm.
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The intensity of irACTH peptide staining within the pars distalis did
not appear different after 96 h of cortisol treatment (Fig.
8D), compared with the
saline-infused animals (Fig. 8B). However, the number of irACTH-positive corticotrophs in the pars distalis was significantly reduced (a decrease of 13.9%) from 218.6 ± 12.5 immunopositive cells per 1.7 × 105
µm2 in the saline controls to
188.2 ± 15.1 immunopositive cells per 1.7 × 105
µm2 after 96 h of cortisol
infusion. There was no diminution in the number of immunopositive cells
in the pars intermedia after 96 h of cortisol infusion (Fig.
8H ) compared with control (Fig. 8F). Adjacent sections were
incubated with antibody preabsorbed with an excess of human
ACTH-(1
24), and there was no staining present (data not shown; see
also Ref. 22).
 |
DISCUSSION |
We have shown that, in the late-gestation ovine fetus, mean plasma
irACTH concentrations rise significantly in response to physiological
elevations in plasma cortisol concentrations. However, this does not
appear to be due to a decrease in cortisol negative feedback, since
plasma free cortisol concentrations remained elevated despite an
increase in plasma CBG levels. The high concentration of plasma free
cortisol suppressed pituitary POMC mRNA levels in both the pars
distalis and pars intermedia, and the number of irACTH immunopositive
cells within the pituitary pars distalis was also reduced after 96 h of
cortisol infusion. However, the number of immunopositive cells within
the pars intermedia did not appear to be appreciably altered in the
presence of elevated circulating free cortisol.
We believe that the increase in plasma irACTH predominantly reflects
changes in ACTH-(1
39). The ACTH antibody used to measure fetal ACTH
concentrations has been validated extensively and does not show
cross-reactivity with the higher molecular weight ACTH-related peptides. We suggest that the increase in irACTH reflects an increase in ACTH output in response to the infusion of cortisol, although we
cannot exclude the possibility that the metabolic clearance rate of
circulating ACTH is reduced by cortisol infusion. Mean fetal arterial
pressure increased by ~7 mmHg within the first 24 h of cortisol
infusion and remained at this level throughout the experiment. This is
similar to the increase reported during a 48-h cortisol infusion to the
midgestation ovine fetus (17). However, the hypertensive effects of
cortisol do not elicit an immediate ACTH response and seem very
unlikely to be causal in the later rise in plasma irACTH
concentrations. Although uterine activity does increase slightly within
the last 24 h of cortisol infusion, this event is preceded by the ACTH
rise; therefore the initial rise in plasma irACTH cannot be labor
induced. The increase in plasma irACTH at 72-96 h may be
associated with an increase in uterine activity; however, there was no
change in fetal arterial PO2, which
indicates that the rise in ACTH is not hypoxia mediated. In addition,
there was no change in the plasma irACTH concentrations of the
saline-treated fetuses, verifying that the rise in ACTH is not an
effect of the sampling protocol employed.
It has previously been reported that a 96-h cortisol infusion to the
ovine fetus at 134 days of gestation affects pulsatility of irACTH,
increasing pulse peak and nadir (3). We examined the maximal and
minimal changes in irACTH and compared these with the mean irACTH
concentration during the control period. The maximal change in irACTH
from the three plasma samples collected each day was significantly
elevated during the cortisol infusion compared with that of control
fetuses. However, we did not employ a frequent sampling protocol
necessary to establish whether the cortisol infusion administered in
these animals altered ACTH pulse frequency over the course of the
experiment.
It has been demonstrated that plasma CBG levels rise within 2-4
days of cortisol infusion (3, 5). In the present study, plasma CBG
levels rose in response to cortisol, becoming significantly greater
than those in controls at 48-72 h of infusion. This was associated
with an increase in hepatic CBG mRNA levels. Low-dose cortisol infusion
to the ovine fetus at 100 days of gestation (for 100 h), or
administration of dexamethasone at 130 days of gestation (for 96 h),
increased CBG biosynthesis and secretion and also altered the pattern
of CBG glycosylation (5, 6). Changes in CBG glycoforms may increase the
half-life of CBG in the circulation, and this may account for the
earlier rise in CBC in plasma than in steady-state levels of hepatic
CBG mRNA determined in the present study.
The percentage of free cortisol in plasma rose within 8 h of
cortisol infusion and then returned to control levels by 72 h of
infusion. However, the absolute concentrations of free cortisol remained elevated throughout the experiment. This suggests that CBG is
effective at maintaining the percentage of free cortisol in fetal
circulation but does not control the absolute concentration of free
cortisol during periods of rapidly increasing plasma cortisol concentrations. This reflects the CBG response to increasing plasma cortisol concentrations in the sheep fetus at term. Plasma CBG and
cortisol concentrations rise in parallel during late gestation, and low
free cortisol concentrations are effectively maintained until the last
5 days of pregnancy (4, 7). Although CBG did not appear to be effective
in decreasing the negative-feedback effects of the rapidly increasing
plasma cortisol concentrations, this does not preclude a role for
circulating CBG in modifying feedback control of the prepartum rise in
plasma ACTH levels. In addition, we showed earlier that the fetal sheep
pituitary synthesizes CBG (7), which could alter local feedback
mechanisms. However, we did not determine changes in pituitary CBG
biosynthesis in the current study.
POMC mRNA levels in the pars intermedia and pars distalis were not
affected by 12 h of cortisol treatment, as has been demonstrated previously (28). However, after 96 h of cortisol infusion, POMC mRNA
levels were suppressed in both the pars intermedia and pars distalis.
In addition, the number of irACTH-positive cells within the pars
distalis was decreased after 96 h of cortisol treatment, suggesting
that the elevated plasma free cortisol concentrations are exerting
negative-feedback effects on the corticotrophs of the pars distalis.
Nonetheless, it is possible that cortisol affects the rate of POMC
translation or ACTH secretion. However, within the pars intermedia
there was no apparent change in irACTH-positive cells or the intensity
of irACTH staining. Pars intermedia corticotrophs from fetal sheep
secrete ACTH-(1
39) in vitro (18). In addition, attenuation of the
endogenous cortisol rise, by bilateral fetal adrenalectomy, does not
alter the basal ACTH-(1
39) output from subsequently cultured pars
intermedia cells (18). The pars intermedia may therefore provide an
additional source of ACTH. In the presence of high plasma
concentrations of cortisol, there may be an alteration of POMC
processing within the pars intermedia, resulting in an increase in
circulating ACTH-(1
39). Alternatively, the pars intermedia may
secrete large molecular weight POMC products into the circulation (34),
which are processed to ACTH-(1
39) under the influence of elevated
cortisol at other sites.
Schwartz et al. (35) have examined the regulation of pituitary ACTH
secretion in vitro and have shown that there are subpopulations of
corticotrophs within the pars distalis which are differentially regulated. These are the CRH-sensitive corticotroph, which secretes ACTH in response to CRH via an increase in adenosine
3',5'-cyclic monophosphate, and the arginine vasopressin
(AVP)-responsive corticotroph, which stimulates ACTH release by a
second pathway likely involving inositol trisphosphate (38). In vitro
studies indicate that glucocorticoids do not inhibit ACTH-(1
39)
secretion and significantly increased ACTH precursor release from the
AVP-responsive corticotrophs (35). A direct effect of cortisol on fetal
corticotrophs has also been described. Antolovich et al. (2)
characterized a change in the morphology of the corticotrophs from
ovine fetuses treated with cortisol from a "fetal-" to an
"adult-type" cell. This morphological change occurs normally
during late gestation but did not occur in adrenalectomized fetuses
(2). Histological maturation of the corticotroph may also be associated
with altered processing of POMC to produce ACTH-(1
39) preferentially
(2). ACTH-(1
39) is the predominant POMC product of the adult
corticotroph (36). In support of this possibility, Brieu and Durand
found that fetal ovine corticotroph cells treated with cortisol (4 days) in vitro decreased the output of total ACTH peptides but
increased the relative proportion of immunoreactive material that
coeluted with ACTH-(1
39) (9) and augmented the release of bioactive ACTH (8). The pituitaries collected for the present study were slow-
frozen for in situ hybridization, and therefore the cellular morphology
could not be examined effectively. It remains possible that the rise in
plasma irACTH in response to the cortisol infusion could be a result of
AVP-stimulated ACTH release and/or a change in the processing
of POMC such that ACTH-(1
39) is preferentially released from POMC
remaining in the anterior pituitary corticotrophs. Alternatively, we
speculate that changes in prohormone convertase activities (39) in the
pars distalis and pars intermedia of the fetal pituitary might lead to
increased proportions of ACTH-(1
39) secreted or that circulating ACTH
might be derived from alternate sources, including the lung and
placenta (15, 16, 24). At present, the effects of cortisol on these
alternative sites of POMC production and processing have yet to be
elucidated.
 |
ACKNOWLEDGEMENTS |
We thank Jac Homan and Drs. Mildred Ramirez, Geert Braems, and Ed
Berdusco for their input and assistance.
 |
FOOTNOTES |
This work was supported by the Medical Research Council (MRC) of Canada
(Group in Fetal and Neonatal Health and Development; G. L. Hammond and J. R. G. Challis). S. G. Matthews was supported by an MRC
fellowship.
Address for reprint requests: T. M. Jeffray, Dept. of Physiology,
Medical Science Bldg., University of Toronto, 1 King's College Circle,
Toronto, ON, Canada M2S 1A8.
Received 16 September 1997; accepted in final form 18 November
1997.
 |
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