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Control of hepatic insulin-like growth factor II gene
expression by thyroid hormones in fetal sheep near term
ALISON J. FORHEAD,1 JUAN LI,1 R. STEWART GILMOUR,2 AND ABIGAIL L. FOWDEN1
Laboratory, University of Cambridge, Cambridge CB2 3EG; and 2Department
of Cellular Physiology, BBSRC Babraham Institute, Cambridge CB2 4AT, United Kingdom
1Physiological
fetus; insulin-like growth factor; thyroid hormones; cortisol
THE INSULIN-LIKE
growth factors (IGFs) are important
regulators of growth in both fetal and neonatal animals, although the mechanisms controlling IGF synthesis differ before and after birth. In many species, IGF-II
is the predominant IGF present in utero and, compared
with expression in the adult animal, is highly abundant
in the fetal circulation and tissues (18, 19, 23). The
importance of IGF-II in the normal development of the
fetus has been demonstrated in transgenic mice in
which the IGF-II gene has been disrupted. Both fetal
and placental size are severely impaired in these
animals, although normal rates of growth are observed
after birth (1, 8). In addition, body weight has been
positively correlated with plasma IGF-II concentrations in sheep fetuses during late gestation (32).
In utero, plasma IGF-II concentrations and IGF-II
gene expression in hepatic and extrahepatic tissues
appear to be independent of pituitary control (21, 30).
In fetal sheep, circulating concentrations of growth
hormone (GH) are high, but hepatic expression of GH
receptor and IGF-I genes remains low until shortly
before birth (3, 27). The ontogenic increase in GHmediated IGF-I production coincides with a decrease in
plasma IGF-II concentrations and downregulation of
IGF-II gene expression in the liver and other fetal
tissues (9, 18, 28). Hence, the transition from fetal to
neonatal life is characterized by a developmental shift
in hepatic IGF synthesis.
In sheep, the maturational switch in hepatic IGF
production from IGF-II to IGF-I and the induction of
hepatic GH receptors depend on the increase in fetal
plasma cortisol concentrations that normally occurs
toward term (27, 28). This prepartum cortisol surge is
also known to be responsible for a number of other maturational events that prepare the sheep fetus for extrauterine life (29). For example, cortisol stimulates the
deiodination of thyroxine (T4 ) to triiodothyronine (T3 )
and thereby causes a prepartum increase in circulating
T3 concentrations that coincides with the decline in
hepatic IGF-II gene expression toward term (14, 15, 28).
Thyroid hormones are known to be essential for
normal growth and development in utero. Hypothyroidism in fetal sheep causes growth retardation and
abnormal maturation of a number of fetal tissues (11,
13, 20). It is also associated with changes in circulating
IGF concentrations in the fetus (21, 30). However, little
is known about the role of thyroid hormones in the
regulation of tissue IGF gene expression in utero,
especially during late gestation when major changes in
IGF mRNA abundance occur as fetal plasma cortisol
concentrations rise.
Therefore, the present study investigated 1) the role
of thyroid hormones in the control of hepatic IGF-II
gene expression during late gestation and 2) whether
the prepartum rise in plasma T3 concentrations may
mediate the maturational action of cortisol on hepatic
IGF-II mRNA abundance. The effects of thyroid hormones on hepatic IGF-II gene expression were examined in sheep fetuses after experimental manipulation
of plasma thyroid hormone concentrations by fetal
thyroidectomy and exogenous hormone infusion.
0193-1849/98 $5.00 Copyright r 1998 the American Physiological Society
E149
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Forhead, Alison J., Juan Li, R. Stewart Gilmour, and
Abigail L. Fowden. Control of hepatic insulin-like growth
factor II gene expression by thyroid hormones in fetal sheep
near term. Am. J. Physiol. 275 (Endocrinol. Metab. 38):
E149–E156, 1998.—The effects of thyroid hormones on hepatic insulin-like growth factor (IGF) II gene expression and
their interaction with cortisol in the ontogenic control of this
gene were investigated in fetal sheep during late gestation
(term 145 6 2 days) and after experimental manipulation of
fetal plasma hormone concentrations. In intact fetuses, a
significant decrease in hepatic IGF-II mRNA abundance was
observed between 127–130 and 142–145 days of gestation,
which coincided with the normal prepartum rise in plasma
cortisol and triiodothyronine (T3 ) concentrations. This ontogenic decline in hepatic IGF-II gene expression was abolished
in fetuses in which the prepartum rise in plasma T3, but not
cortisol, was prevented by fetal thyroidectomy. At 127–130
days, downregulation of hepatic IGF-II mRNA abundance
was induced prematurely in intact fetuses by an infusion of
cortisol for 5 days (2–3 mg · kg21 · day21 iv). Plasma concentrations of cortisol and T3 in the cortisol-infused intact fetuses
were increased to values seen close to term. Similar findings
were observed in thyroidectomized fetuses, in which, despite
thyroidectomy, cortisol infusion significantly increased plasma
T3 concentrations and caused a premature decrease in hepatic IGF-II mRNA levels. However, in intact fetuses at
127–130 days, the increasing of T3 concentrations alone by
exogenous T3 infusion (8–12 µg · kg21 · day21 iv for 5 days) had
no effect on hepatic IGF-II mRNA levels. Overall, a decrease
in hepatic IGF-II mRNA abundance was only observed in
fetuses in which there were concurrent increases in plasma
cortisol and T3 concentrations. When observations from all
fetuses were considered, irrespective of gestational age or
treatment, hepatic IGF-II mRNA levels were negatively
correlated with plasma cortisol and T3 but not thyroxine
concentrations. Partial correlation analysis of hepatic IGF-II,
cortisol, and T3 values showed that the plasma concentration
of cortisol in the fetus had the predominant effect on hepatic
IGF-II mRNA abundance. These findings show that T3 may
mediate, in part, the maturational effects of cortisol on
hepatic IGF-II gene expression but that it is ineffective
without a concomitant rise in fetal plasma cortisol. Hence,
increased concentrations of both cortisol and T3 appear
necessary to induce downregulation of hepatic IGF-II mRNA
abundance in fetal sheep close to term.
E150
HORMONAL CONTROL OF HEPATIC IGF-II
MATERIALS AND METHODS
Table 1. Number and gestational ages of fetuses used
in different experimental groups
Gestational Age, days
Treatment
At
thyroidectomy
Intact and
untreated
Intact and
cortisol
infusion
Intact and
T3 infusion
Thyroidectomy
and untreated
Thyroidectomy
and cortisol
infusion
At
catheterization
115–120 (n 5 3) 127–130
142–145
6
4
115–120
127–130
4
115–120
127–130
5
127–130
142–145
4
4
127–130
4
105–110
105–110
105–110
Number
At tissue
of
collection Fetuses
115–120
Value in parentheses is no. of fetuses catheterized at 115–120 days.
T3 , triiodothyronine.
IN FETAL SHEEP
ate prepartum period. Of the remaining untreated fetuses,
three intact and four TX fetuses were delivered at 127–130
days, and four intact and four TX fetuses were delivered at
142–145 days of gestation.
All fetuses were delivered by cesarean section under general anesthesia (20 mg/kg iv pentobarbital sodium). At delivery, blood samples were taken by venipuncture of the umbilical artery. All blood samples obtained during the study were
placed into EDTA-containing tubes and centrifuged for 5 min
at 1,000 g and 4°C; the plasma aliquots were stored at 220°C
until analysis. A number of tissues were collected from the
fetuses immediately after the administration of a lethal dose
of pentobarbital sodium (200 mg/kg). Samples of liver were
snap-frozen in liquid nitrogen and stored at 280°C until
analysis. At delivery, there was no evidence of thyroidal
remnants in any of the TX fetuses.
Biochemical analysis. Plasma cortisol concentrations were
measured by radioimmunoassay validated for use with ovine
plasma as described previously (37). The lower limit of
detection was 40–80 pmol/l, and the interassay coefficient of
variation was 11%. Plasma T3 and T4 concentrations were
also measured by radioimmunoassay, using a commercial kit
validated for ovine plasma (13) (ICN Biomedicals, Thame,
UK). The lower limits of detection were 0.21 nmol/l for T3 and
8.8 nmol/l for T4. The interassay coefficient of variation was
10% for both assays.
Tissue RNA isolation and RNase protection assay. Total
RNA was extracted from 1 g of frozen tissue, using the
guanidine thiocyanate method of Chomczynski and Sacchi
(6), and was quantified by absorbance at 260 nm (1 optical
density 5 35 µg/ml). To check the equivalence of the RNA
samples, total poly(A)1 was also measured as described
previously (39). When RNA samples from all tissues were
considered, there was a constant relationship between absorbance and total poly(A)1 content. The RNase protection assay
was carried out on 50-µg samples of total RNA with the use of
ovine IGF-II riboprobe specific for coding exon 8 as described
previously (28). The relative intensities of the protected band
on the X-ray film were quantified by densitometry and normalized, using a number of control samples run on all gels.
Statistical analysis. All data are presented as mean
values 6 SE. Significant differences in the measurements
made between the different groups of fetuses were assessed
by unpaired t-test. Within each group of fetuses infused with
either cortisol or T3, paired t-tests were used to compare the
values observed before the infusion began and 5 days later.
Relationships between the variables measured were determined by linear regression and partial correlation analyses
(41); plasma hormone concentrations that were below the
limit of assay detection were assigned a value of 1 in
regression analyses. Differences for which P , 0.05 were
regarded as significant.
RESULTS
Ontogeny of hepatic IGF-II gene expression in intact
and TX fetuses. The normal ontogenic decline in hepatic IGF-II mRNA abundance was abolished when the
immediate prepartum rise in plasma T3 but not cortisol
concentrations was prevented by fetal TX (Fig. 1).
At 127–130 days of gestation, there was no significant difference in hepatic IGF-II mRNA levels between
TX and intact fetuses (Fig. 1 and Table 2A). Plasma
cortisol and T3 concentrations were also similar between the two groups of fetuses at this gestational age
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Animals. A total of 31 Welsh Mountain sheep fetuses of
known gestational age were used in this study. All but two of
the fetuses were twins. The ewes were housed within the
laboratory animal house in individual pens and were maintained on 200 g/day concentrates, with free access to hay,
water, and a salt-lick block. Food but not water was withheld
for 18–24 h before surgery. Table 1 shows the number and
gestational ages of the fetuses used in each experimental
group of the study.
Surgical procedures. All surgical operations were performed under halothane anesthesia (1.5% in O2-N2O) with
positive pressure ventilation. Between 105 and 110 days of
gestation (term 145 6 2 days), 12 fetuses were thyroidectomized (TX) in utero with the use of surgical techniques
described previously (20). Four of these TX fetuses were
catheterized in a second operation at 115–120 days along
with 12 of the intact fetuses. Intravascular catheters were
inserted into the femoral artery and a branch of the femoral
vein in the fetuses and into the maternal femoral artery as
described previously (7). All catheters were exteriorized
through the flank of the ewe and secured in a plastic pouch
sutured to the skin. The catheters were flushed daily with
heparinized saline solution [100 IU heparin/ml in 0.9%
(wt/vol) saline] from the day after surgery. At surgery, all
fetuses were administered intravenously 100 mg of ampicillin
(Penbritin, Beecham Animal Health, Brentford, UK) and 2
mg of gentamycin (Frangen-100, Biovet, Mullingar, UK). The
ewes received antibiotic intramuscularly (900 mg of procaine
penicillin; Depocillin, Mycofarm, Cambridge, UK) on the day
of surgery and for 3 days thereafter. Normal feeding patterns
were restored within 24 h of the operation.
Experimental procedures. Blood samples of 2 ml were taken
daily from the catheterized fetuses and ewes to monitor fetal
well-being and blood gas status and to determine plasma
hormone concentrations.
Four intact and four TX fetuses were infused with cortisol
(2–3 mg · kg21 · day21 iv; Efcortelan, Glaxo, Ware, UK) for 5
days before delivery for tissue collection at 127–130 days. In
addition, eight intact fetuses were infused with either T3
(8–12 µg · kg21 · day21 iv, n 5 5; Sigma, Poole, UK) or saline
(n 5 3) for 5 days before delivery at 127–130 days. The doses
of exogenous cortisol and T3 infused were calculated to mimic
the plasma concentrations normally observed in the immedi-
MRNA
HORMONAL CONTROL OF HEPATIC IGF-II
MRNA
E151
IN FETAL SHEEP
(Table 2A). Plasma T4 concentrations were undetectable in the TX fetuses and were significantly lower than
those observed in the intact fetuses at 127–130 days
(P , 0.01, Table 2A).
Between 127–130 and 142–145 days, a significant
decrease in IGF-II mRNA abundance was seen in the
intact (P , 0.001, Fig. 1) but not TX fetuses. At
142–145 days, hepatic IGF-II mRNA levels in the TX
fetuses were similar to those seen in TX fetuses at
127–130 days and were significantly greater than those
observed in the intact fetuses (Fig. 1 and Table 2A).
Between 127–130 and 142–145 days, plasma cortisol
concentrations significantly increased in both groups of
fetuses (P , 0.05 in both cases, Table 2A), but a
significant concomitant increase in plasma T3 concentrations was only observed in the intact fetuses (P , 0.01,
Table 2. Plasma concentrations of cortisol, T3 , and T4 and hepatic IGF-II mRNA levels
Treatment
Cortisol, nmol/l
Gestational Age at
Delivery, days
Preinfusion
Delivery
T3 , nmol/l
T4 , nmol/l
Delivery
IGF-II mRNA at Delivery,
arbitrary units
160.0 6 26.5
NDa
125.4 6 8.6
ND
46.5 6 3.1
39.2 6 3.0
8.0 6 3.1b
37.2 6 3.9
ND
ND
160.0 6 26.5
1.60 6 0.40b,e 159.0 6 20.8 133.3 6 7.4
ND
ND
0.64 6 0.07b,f
ND
ND
46.5 6 3.1
19.3 6 5.4b
39.2 6 3.0
8.7 6 2.9d
ND
ND
160.0 6 26.5
0.85 6 0.04b,f 130.6 6 12.6 107.6 6 10.6
46.5 6 3.1
49.5 6 8.0
Preinfusion
Delivery
Preinfusion
A
32.6 6 2.5
22.3 6 2.5
161.4 6 38.6a
172.1 6 42.2c
Intact
TX
Intact
TX
127–130
127–130
142–145
142–145
Intact
Intact 1 cortisol
TX
TX 1 cortisol
127–130
127–130
127–130
127–130
32.6 6 2.5
28.7 6 3.9 175.2 6 32.6b,e
22.3 6 2.5
25.7 6 6.1 189.2 6 14.1d,e
Intact
Intact 1 T3
127–130
127–130
29.0 6 4.1
ND
ND
1.00 6 0.24a
ND
B
ND
C
32.6 6 2.5
33.7 6 4.7
Values are mean 6 SE plasma concentrations of cortisol, T3 , and thyroxine (T4 ) and hepatic insulin-like growth factor (IGF) II mRNA levels
in intact and thyroidectomized (TX) fetuses at 127–130 and 142–145 days (A), intact and TX fetuses infused with cortisol for 5 days (B), and
intact fetuses infused with T3 for 5 days (C ). ND, not detectable (below lower limit of assay detection). a P , 0.05 and b P , 0.005, significantly
different from intact fetuses at 127–130 days, unpaired t-test. c P , 0.05 and d P , 0.005, significantly different from TX fetuses at 127–130
days, unpaired t-test. e P , 0.05 and f P , 0.005, significantly different from preinfusion values, paired t-test.
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Fig. 1. Autoradiogram analysis (A) and mean 6
SE relative values (B) of hepatic insulin-like
growth factor (IGF) II mRNA abundance in intact
and thyroidectomized (TX) sheep fetuses at 127–
130 and 142–145 days (d) of gestation. * Significantly different from intact fetuses at 127–130
days, P , 0.001. o Significantly different from
intact fetuses at 142–145 days, P , 0.001.
E152
HORMONAL CONTROL OF HEPATIC IGF-II
Fig. 2. Autoradiogram analysis (A) and mean 6
SE relative values (B) of hepatic IGF-II mRNA
abundance in intact and TX sheep fetuses at
127–130 days and after a 5-day infusion of cortisol. * Significantly different from untreated and
saline-infused intact fetuses at 127–130 days,
P , 0.005. † Significantly different from untreated TX fetuses at 127–130 days, P , 0.001.
IN FETAL SHEEP
Over the period of cortisol infusion, plasma T3 concentrations significantly increased in both intact (P , 0.05)
and TX fetuses (P , 0.005, Table 2B). At delivery at
127–130 days, concentrations of T3 in the cortisolinfused intact and TX fetuses were significantly higher
than those measured in their respective groups of
control fetuses (P , 0.005 in both cases, Table 2B) and
were similar to those observed in intact fetuses at
142–145 days (Table 2, A and B). Plasma T4 concentrations were unaffected by cortisol infusion in both groups
of fetuses (Table 2B).
Effect of T3 infusion on hepatic IGF-II gene expression
in intact fetuses. Hepatic IGF-II mRNA levels at 127–
130 days were unaffected when fetal plasma T3, but not
cortisol, concentrations were raised to values seen near
term by an exogenous infusion of T3 for 5 days before
delivery (Fig. 3).
No significant difference in hepatic IGF-II mRNA
abundance was observed between T3-infused and control intact fetuses at 127–130 days (Fig. 3 and Table
2C). Infusion of T3 significantly increased plasma T3
concentrations (P , 0.0001, Table 2C). At delivery at
127–130 days, plasma T3 concentrations in the T3infused fetuses were significantly greater than those
observed in control intact fetuses (P , 0.0001, Table
2C) and were similar to the concentrations seen in
intact fetuses at 142–145 days of gestation (Table 2, A
and C). Plasma cortisol and T4 concentrations in the
T3-infused fetuses at 127–130 days were similar to
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Table 2A). No gestational change in plasma T4 concentrations was identified in either group of fetuses;
plasma T4 remained undetectable in the TX fetuses at
142–145 days (Table 2A).
Effect of cortisol infusion on hepatic IGF-II gene
expression in intact and TX fetuses. Hepatic IGF-II
mRNA abundance was reduced in both intact and TX
fetuses at 127–130 days when fetal plasma cortisol
concentrations were increased to values seen near term
by an exogenous infusion of cortisol for 5 days (Fig. 2).
At 127–130 days, hepatic IGF-II mRNA abundance
in the cortisol-infused intact fetuses was significantly
lower than that observed in the control intact fetuses
(P , 0.005, Fig. 2 and Table 2B). Likewise, hepatic
IGF-II mRNA abundance in the TX fetuses infused
with cortisol was significantly lower than that seen in
the untreated TX fetuses (P , 0.001, Fig. 2 and Table
2B). There was no significant difference in hepatic
IGF-II mRNA levels between intact and TX fetuses
infused with cortisol (Fig. 2).
Exogenous cortisol infusion significantly increased
plasma cortisol concentrations in intact and TX fetuses
to a similar extent (P , 0.05 in both cases, Table 2B). At
delivery, plasma cortisol concentrations in the cortisolinfused intact and TX fetuses were significantly greater
than those observed in their respective groups of control fetuses at 127–130 days (P , 0.005 in both cases,
Table 2B) and resembled values seen in intact and TX
fetuses at 142–145 days of gestation (Table 2, A and B).
MRNA
HORMONAL CONTROL OF HEPATIC IGF-II
MRNA
IN FETAL SHEEP
E153
DISCUSSION
those seen in control intact fetuses of the same gestational age (Table 2C).
Relationship between hepatic IGF-II gene expression
and plasma cortisol and thyroid hormone concentrations. When the data from all of the fetuses were
combined, irrespective of treatment or gestational age,
a significant inverse relationship was identified between hepatic IGF-II mRNA abundance and plasma
cortisol concentrations (r 5 20.680, n 5 30, P ,
0.0001). In addition, hepatic IGF-II mRNA levels were
inversely correlated with plasma T3 (r 5 20.367, n 5
31, P , 0.05) but not T4 concentrations (r 5 20.174, n 5
29, P . 0.05). Partial correlation analysis showed that
the plasma concentration of cortisol in the fetus was a
more important determinant of hepatic IGF-II mRNA
abundance (r 5 20.610, n 5 30, P , 0.05) than the
plasma T3 concentration (r 5 20.019, n 5 30, P . 0.05).
A significant positive relationship was also observed
between plasma concentrations of cortisol and T3 in the
fetuses (r 5 0.539, n 5 30, P , 0.005).
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Fig. 3. Autoradiogram analysis (A) and mean 6 SE relative values
(B) of hepatic IGF-II mRNA abundance in intact sheep fetuses at
127–130 days and after a 5-day infusion of triiodothyronine (T3 ).
The present study demonstrates that thyroid hormones have an important role in the control of hepatic
IGF-II gene expression in fetal sheep close to term.
Fetal thyroidectomy abolished the decline in hepatic
IGF-II mRNA abundance that normally occurs toward
term in this species. It also prevented the normal
prepartum rise in fetal plasma T3, but not cortisol,
concentrations. Downregulation of the hepatic IGF-II
gene toward term has been shown previously to be
dependent on the prepartum cortisol surge (28). Hepatic IGF-II mRNA abundance was maintained when
the cortisol surge was prevented by fetal adrenalectomy and was reduced prematurely when cortisol concentrations were raised to values seen at term (28).
However, adrenalectomy also prevented the prepartum
rise in plasma T3, whereas cortisol infusion increased
T3 concentrations by stimulating tissue type I 58monodeiodinase activity and the production of T3 from
T4 (40, 42). These observations indicate that T3 may
mediate, at least in part, the maturational effects of
cortisol on hepatic IGF-II gene expression in utero.
Certainly, in the present study, suppression of hepatic
IGF-II mRNA abundance only occurred in fetuses in
which there were concurrent increases in plasma T3
and cortisol. Raising plasma T3 concentrations alone in
immature fetuses with low circulating cortisol had no
influence on hepatic IGF-II mRNA abundance, nor did
the prepartum cortisol surge in hypothyroid fetuses. In
addition, abolition of the rises in both plasma T3 and
cortisol by fetal hypophysectomy has been shown to
prevent the normal prepartum decline in plasma IGF-II
concentrations (18, 30). Therefore, simultaneous increases in plasma cortisol and T3 appear to be needed to
downregulate IGF-II gene expression in fetal ovine
liver near term.
Although hepatic IGF-II mRNA abundance may be
regulated by the increase in plasma T3 close to term, it
does not appear to be modulated by thyroid hormones
earlier in gestation. In the present study, hypothyroidism induced by thyroidectomy had little influence on
hepatic IGF-II mRNA levels before the immediate
prepartum period. Similarly, tissue content and plasma
concentrations of IGF-II were normal during late gestation in sheep and pig fetuses made hypothyroid by fetal
hypophysectomy (21, 30). Plasma T4, therefore, appears to have little, if any, direct role in the control of
hepatic IGF-II gene expression in utero. Indeed, no
correlation was observed between hepatic IGF-II mRNA
levels and plasma T4 in fetuses in the present study.
Regulation of hepatic IGF-II gene expression close to
term, therefore, does appear to depend primarily on the
cortisol-induced rise in plasma T3. These observations
are consistent with previous studies in neonatal rats
that show that maturational changes in hepatic IGF-II
synthesis and plasma IGF-II concentrations are associated with an increase in circulating corticosterone
and are severely attenuated by hypothyroidism (4, 16,
24, 31).
E154
HORMONAL CONTROL OF HEPATIC IGF-II
IN FETAL SHEEP
and brain of fetal rats (38). In the present study, it is
possible that exogenous cortisol infusion increased
plasma T3 concentrations in TX fetuses by desulfating
T3S in fetal tissues. Indeed, the cortisol-induced rise in
plasma T3 observed in intact fetuses in this and previous studies (14, 15) may have originated from activation of both sulfatase and 58-monodeiodinase enzymes.
The endogenous cortisol surge close to term, however,
did not appear to stimulate T3 production in TX fetuses
at 142–145 days. Plasma T3 concentrations in TX
fetuses did not show any ontogenic change and remained undetectable at term. The inconsistency between the effects of exogenous and endogenous cortisol
on circulating T3 concentrations in TX fetuses may
have been due to the longer interval between thyroidectomy and tissue collection in the older group of animals
and, hence, a greater degree of T3S clearance (43).
Although T3S concentrations were not measured in the
present study, the absence of a cortisol-induced rise in
plasma T3 in the TX fetuses close to term may have
reflected deficiency of not only T4 but also T3S.
Regardless of the source of T3, an increment in its
bioavailability appears to be essential for the cortisolinduced downregulation of hepatic IGF-II gene expression. However, the mechanisms by which these hormones affect IGF-II mRNA abundance remain unknown.
In the present study, the ovine IGF-II riboprobe used
was specific to the main coding exon 8 (28), and,
therefore, IGF-II mRNA levels measured represented
total IGF-II gene expression in the fetal liver. In fetal
and neonatal sheep, at least three leader exons, 5, 6,
and 7, of the IGF-II gene are known to be alternatively
spliced to the main coding exon, and, hence, a number
of mRNA transcripts are derived from the single gene
(25). Expression of each of these transcripts may be
under developmental and tissue-specific control. Previous studies have demonstrated that cortisol downregulates IGF-II mRNA abundance in the liver of the sheep
fetus specifically by suppressing expression of leader
exon 7 (25). A region of 172 bases downstream of the
first transcription site for leader exon 7 has been shown
to respond to cortisol, but no consensus sequences for
repressive glucocorticoid or thyroid hormone response
elements have yet been identified in the regulatory
regions of the IGF-II gene (25). Alternatively, cortisol
and thyroid hormones may act indirectly on the IGF-II
gene via transcription factors and their binding to DNA
(5, 22).
The actions of cortisol and T3 in suppressing hepatic
IGF-II gene expression close to term may have major
implications for growth and development in utero.
They may provide an explanation for the decline in
growth rate that occurs toward term and in response to
cortisol infusion earlier in gestation (12). They may also
have an important role in the prepartum maturation of
the fetal liver (11). Furthermore, downregulation of
hepatic IGF-II gene expression close to term forms part
of a more general developmental change in the somatotrophic axis. At birth, IGF synthesis switches from a
local GH-independent production of predominantly
IGF-II in utero to the adult GH-dependent production
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The ineffectiveness of exogenous T3 alone in downregulating hepatic IGF-II gene expression at 127–130
days may have been due to the relatively low number of
nuclear thyroid hormone receptors present in fetal
tissues at this gestational age (34). In sheep and pig
fetuses, hepatic thyroid hormone binding increases
progressively from 80 days of gestation to peak at birth
in parallel with the rise in fetal plasma cortisol (10, 34).
This ontogenic increase in hepatic thyroid hormone
binding may contribute to the effects of the endogenous
rise in plasma T3 on hepatic IGF-II gene expression
close to term. However, if T3 mediates the effects of
cortisol, the ontogenic change in thyroid hormone receptor density might be expected to make exogenous
cortisol less effective at suppressing hepatic IGF-II
mRNA abundance at 127–130 days than the endogenous rise in cortisol at term. However, in the present
study, hepatic IGF-II mRNA levels in the cortisolinfused intact and TX fetuses were similar to those
seen in intact fetuses at 142–145 days. Because the
ontogeny of hepatic thyroid hormone binding is not
affected by fetal thyroidectomy (34), these observations
suggest that cortisol may act indirectly to suppress
hepatic IGF-II gene expression not only via the production of T3 but also by inducing thyroid hormone receptors in the fetal liver. This latter action of cortisol is in
keeping with its other known maturational effects on
hormone receptors in the ovine fetal liver (2, 27).
It was not possible to investigate the effect of increasing concentrations of cortisol early in gestation in the
absence of a concomitant rise in plasma T3 because
plasma T3 concentrations increased in response to
exogenous cortisol infusion in the TX fetuses. This
observation was surprising. In all TX fetuses, there was
no evidence of thyroidal tissue at delivery, and plasma
concentrations of T4 were below the limit of detection of
the radioimmunoassay. One source of plasma T3 in TX
fetuses may be the sulfated forms of thyroid hormones
that are abundant in the circulation of the sheep fetus
and can account for up to 80% of T4 metabolism and
clearance (35). In intact fetuses, plasma concentrations
of T4 sulfate (T4S), reverse T3 sulfate (rT3S), and T3
sulfate (T3S) peak at 125–130 days and decrease thereafter when T3 production becomes the major route of T4
metabolism toward term (35). Although the sulfated
thyroid hormones do not bind to nuclear thyroid hormone receptors and therefore have no biological activity, they may contribute to plasma and tissue concentrations of T3, especially during fetal hypothyroidism (33,
43). Thyroidectomy in the sheep fetus at 110–113 days
has been shown previously to cause a marked reduction
in plasma concentrations of thyroid hormones and T4S
and rT3S (43). Plasma T3S concentrations, however, are
maintained for at least 2 wk after the removal of the
thyroid glands because of downregulation of type I
monodeiodinase enzyme activity (36, 43). Therefore,
slow clearance of T3S in the TX fetuses may have
provided a supply of T3 to fetal tissues after thyroidectomy. Although the site and regulation of desulfation of
T3S to T3 in the sheep fetus are unknown, sulfatase
activity has been reported in microsomes from the liver
MRNA
HORMONAL CONTROL OF HEPATIC IGF-II
of endocrine IGF-I (17). Many of the changes in gene
expression associated with this transition have now
been shown to be cortisol dependent, but the extent to
which they may also rely on the concomitant change in
plasma T3 remains unknown. Recent preliminary findings suggest that the cortisol-induced upregulation of
hepatic GH receptor gene expression in utero may be
influenced by the thyroid hormones (26). Inappropriate
activity of the fetal thyroid and adrenal cortex may,
therefore, lead to developmental abnormalities in the
somatotrophic axis and IGF production with adverse
consequences for growth both before and after birth.
Received 24 December 1997; accepted in final form 13 April 1998.
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