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Research Communications |
Department of Physiology, University of Cambridge, Cambridge CB2 3EG; and
a Department of Cellular Physiology, The Babraham Institute, Babraham, Cambridge CB2 4AT
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: fetus • GHR • fetal plasma cortisol • GH binding • riboprobe • insulin-like growth factor • glucocorticoid response element
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In adults, the GH receptor (GHR) is encoded by a single gene from which multiple mRNA transcripts are derived (8) . Variations in GHR mRNA have been observed in a wide variety of species including primates, rodents, and ungulates 9-14) . This heterogeneity of GHR gene expression occurs primarily in the 5' untranslated region of the GHR mRNA, though deletions of coding exon 3 have also been reported in the human (8) . In the sheep, two untranslated leader exons (1A and 1B) are known to be alternatively spliced to the coding exons whereas, in the human, at least eight variant GHR mRNA transcripts have been identified (9 , 13,15 ). Postnatally, the variant GHR mRNA transcripts have been shown to be differentially regulated in both a developmental and tissue-specific manner 9-15) . Variants that are expressed only in the liver have been identified in several species (12 , 16 , 17 ). In sheep, for instance, it is leader exon 1A expression, which is liver specific (12) .
Much less is known about GHR gene expression in the fetus. Using immunohistochemistry, Northern blotting, and GH binding to isolated membrane preparations, GH receptors have been detected in fetal tissues of a number of species from early in gestation (11 , 13 , 17-20 ). However, their abundance varies widely between tissues and is low in fetal liver compared with postnatal values (13 , 18 , 20 ). Variant forms of GHR mRNA have also been identified in fetal liver from sheep, humans, and rabbits (13 , 16 , 21-23 ). Using probes to GHR coding sequences, up-regulation of hepatic GHR gene expression has been observed in fetal sheep and pigs close to term 24-26) . These changes closely parallel the normal prepartum rise in fetal plasma cortisol and coincide with an increase in GH binding to hepatic cell membranes (5 , 7 , 27 ). In fetal sheep, total hepatic GHR mRNA is known to be regulated by plasma cortisol in utero (24) , but little is known about the effects of cortisol on the transcript composition of the GHR mRNA. Hence, in the present study, expression of the adult liver-specific exon 1A containing transcript of the GHR gene was examined in fetal ovine liver during late gestation and after experimental manipulation of fetal plasma cortisol by fetal adrenalectomy and exogenous cortisol infusion.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Surgical procedures
Under halothane anesthesia (1.5% in
O2/N2O), one of two procedures was carried out
using the surgical techniques described previously: 1)
intravascular catheterization of the intact fetus and ewe
(28)
or 2) fetal adrenalectomy with or without
catheterization of the fetus and ewe (24)
. The number of
animals in each group and the gestational ages of the fetuses at
operation are shown in Table 1
(normal term 145 ±2 days).
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Experimental procedures
Blood samples were taken daily from all the catheterized fetuses
to monitor fetal well-being. At least 5 days after surgery, 15 intact
twin fetuses were infused intravenously with either cortisol
(n=7, 2–3
mg ·kg-1 ·day-1 EF-Cortelan,
Glaxo plc, U.K.), or saline (n=7, 3 ml/day) for 5 days
before delivery. The infusion began either at 105–107 days
(n=3 pairs of twins) or 122–125 days of gestation
(n=4 pairs of twins). The fetus to be infused with cortisol
was chosen randomly. Similarly, continuous infusions of cortisol or
saline (n=4 for each group, as above) were given to eight
adrenalectomized fetuses for 5 days beginning at 122–126 days. Tissues
were collected at the end of the infusion at either 110–112 days or
127–131 days (Table 1)
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Autopsy and sample collection
All surgically treated fetuses (regardless of previous
treatment) and 11 additional fetuses that were not treated surgically
were delivered by Caesarean section under sodium pentobarbitone
anesthesia (6% w/v NaCl 20 mg/kg i.v.). Details of the numbers and age
of the fetuses at delivery are shown in Table 1
. Blood samples were
taken from the fetus at the time of delivery, either through the
indwelling catheter or by venipuncture from the umbilical artery.
Samples of liver were collected from the fetuses after administering a
lethal dose of anesthetic (200 mg/kg i.v. Na pentobarbitone). The
tissue was frozen immediately in liquid nitrogen and stored at
-80°C. All blood samples were centrifuged at 4°C and the plasma
stored at -20°C until required for analysis. There were no adrenal
remnants in any of the adrenalectomized fetuses at autopsy.
Biochemical analyses
Plasma cortisol concentration
The arterial plasma concentration of cortisol was measured by
radioimmunoassay validated for ovine plasma as described previously
(24)
. The interassay coefficients of variation was 10%
and the minimum detectable quantity of hormone was 10–30 pg/tube.
RNA isolation
Total RNA was isolated from 1 g portion of frozen tissues
using the guanidinium thiocyanate method of Chomczynski and Sacchi
(29)
. Total RNA was quantified by absorbency at 260 nm (1
OD = 40 µg). To check equivalence of RNA samples, total poly
(A)+ content was also measured and quantified using an
image analyzer, as described previously (30)
. A constant
relation between OD and poly (A)+ content was found for RNA
from all tissues: the mean (±SEM) intensity of the poly
(A)+ RNA hybridization signal from liver of intact fetuses
at 143–145 days (42.5 ±2.0 arbitrary units, n=4) was not
significantly different from that found earlier in gestation (41.5
±2.0, n=4) or from the values observed after fetal
adrenalectomy (41.0 ±1.5, n=4) and cortisol infusion (39.0
±2.0, n=4).
Construction of riboprobe
A riboprobe containing exons 1A, 2, and 3 of the ovine growth
hormone receptor mRNA was constructed by polymerase chain reaction
(PCR), using oligonucleotide primers as follows: the
5'-ATCT(AAGCTT)CAGCCTCTGTTTCAGGA-3', comprising a HindIII
recognition site (in brackets) and located in the leader exon 1A region
between nucleotides 172 and 188 of the published sequence
(13)
. The 3' primer was
5'-ATTC(GAATTC)TGTCTCTAGGCCTGGAT-3', comprising an EcoRI recognition
site linked to the complement of the DNA sequence of coding region exon
3 of the ovine GHR gene. Using these primers, PCR was performed on cDNA
synthesized from total RNA of liver from an adult sheep, as described
previously (30)
. The product of the 201 bp fragment was
digested with EcoRI and HindIII, cloned into Bluescript KS (Strategene
Ltd., Cambridge, U.K.), and the sequence was verified by
double-stranded sequencing. The plasmid DNA was linearized by HindIII
digestion and used as a template to generate antisense riboprobe in an
in vitro transcription system by using T7 RNA polymerase.
RNase protection assay
The riboprobe was synthesized in the presence of
-32P-UTP (ICN, Irvine, Calif.) to yield a labeled
antisense RNA probe and RNase protection assays were performed on
either 25 or 50 µg of individual liver RNA samples, exactly as
described before (30)
. Protected fragments were separated
on 6% polyacrylamide sequencing gels; the dried gels were exposed to
X-ray film (Kodak, Cambridge, U.K.) and the intensities of the
protected bands quantified using an image analyzer (Seescan Imaging,
Cambridge, U.K.). To allow data from different experiments to be
combined, a standard reference sample was included in each gel and used
to normalize values from separate gels.
Statistical analysis
Means and standard errors are given throughout and statistical
analysis were made according to the methods of Armitage
(31)
. Statistical significance was assessed by one way
ANOVA, paired and unpaired t test with Fisher's test.
Undetectable values for exon 1A expression were assigned a value of
0.05 for the purposes of statistical analyses. Probabilities of less
than 5% were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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At all gestational ages studied, the predominant GHR mRNA transcripts were those derived from leader exons other than 1A (Fig. 1) . Leader exon 1A-containing GHR mRNA was not detectable before 130 days and first appeared in fetal liver at about 138 days of gestation (Fig. 1) . Thereafter, expression of this leader exon rose to account for 30.0 ±7.6% (n=4) of the total GHR mRNA at term (Fig. 2 ). However, even at 143–145 days, abundance of the leader exon 1A-containing transcripts was 10-fold less than seen in adult liver (Fig. 1) . Levels of GHR mRNA derived from the other leader exons also increased in fetal liver toward term (Fig. 1) ; mean abundance of the 146 base band at 143–145 days was similar to that in adult liver (Fig. 1) and significantly higher than the values observed earlier in gestation (Fig. 2) . Up-regulation of GHR gene expression from both the 1A and other leader exons closely paralleled the normal rise in fetal plasma cortisol concentrations toward term (Fig. 2) .
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Manipulation of fetal plasma cortisol
Fetal adrenalectomy
Fetal adrenalectomy abolished the prepartum rise in fetal plasma
cortisol (Table 2
) and prevented activation of leader exon 1A-derived GHR mRNA
expression (Fig. 3a
). At 143–145 days, the mean plasma cortisol in
adrenalectomized fetuses was significantly less than that in intact
fetuses of the same gestational age and was similar to the values
observed in intact fetuses before 130 days (Table 2)
. In contrast to
intact fetuses, no leader exon 1A-derived GHR mRNA was detected in
liver from adrenalectomized fetuses at 143–145 days (Fig. 3a
) nor was there any prepartum increase in GHR mRNA derived
from the other leader exons in the adrenalectomized fetuses (Fig. 3a
). In adrenalectomized fetuses at 143–145 days, the mean
abundance of GHR mRNA derived from the other leader exons was similar
to that in intact and adrenalectomized fetuses at 127–131 days and
significantly less than the value observed in intact fetuses of the
same gestational age (Table 2)
.
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Fetal cortisol infusion
At both 110–112 days and 127–130 days, cortisol infusion into
intact fetuses for 5 days raised fetal plasma cortisol at delivery to
values similar to those seen in intact fetuses at term (Table 2)
.
Expression of GHR leader exon 1A was also activated in the liver of all
cortisol infused intact fetuses irrespective of gestational age,
although the abundance of the exon 1A-derived GHR mRNA appeared to be
less in cortisol infused fetuses than in intact fetuses at term with
similar plasma cortisol levels (Fig. 3a, b, d
, Table 2
). The
abundance of GHR mRNA derived from the other leader exons was also
increased by cortisol infusion in the intact fetuses (Fig. 3b, d
). Mean levels of hepatic GHR mRNA derived from the other leader
exons in cortisol infused fetuses at 110–112 days and 127–130 days
were similar to those seen in intact fetuses at term and were
significantly greater than the values observed in the corresponding
groups of saline-infused intact fetuses (Table 2)
. Cortisol infusion
had similar effects on fetal plasma cortisol and hepatic GHR mRNA
abundance in the adrenalectomized fetuses at 128–131 days (Fig. 3
,
Table 2
). Expression of GHR mRNA containing leader exon 1A was
activated by cortisol infusion in adrenalectomized fetuses (Fig. 3c
). Similarly, the abundance of GHR mRNA transcripts
derived from the other leader exons was significantly greater in
cortisol- than saline-infused adrenalectomized fetuses at 128–131 days
of gestation (Table 2)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cortisol also up-regulated hepatic GHR gene expression from other leader exons in the sheep fetus. The normal prepartum increment in GHR mRNA derived from these other leader exons did not occur after fetal adrenalectomy and could be mimicked earlier in gestation by fetal cortisol infusion. In fact, the transcripts derived from the alternate leader exons accounted for the major part of the increment in hepatic GHR mRNA observed toward term and in response to cortisol infusion. Preliminary observations using an exon 1B-specific riboprobe suggest that the GHR mRNA detected in fetal liver is derived predominantly from exon 1B throughout late gestation (J. Li, R. S. Gilmour, and A. L. Fowden, unpublished observations). Consequently, the prepartum increase in hepatic GHR gene expression observed in utero in previous studies using GHR coding sequence probes (13 , 21 , 24 ) probably reflects enhanced expression from both the 1A and 1B leader exons. Certainly, the current findings are consistent with previous observations that up-regulation of GHR gene expression toward term may involve two GHR mRNA transcripts differing upstream from the first coding exon (13) .
Similar gestational changes in hepatic GHR gene expression have been observed in other species. In the human, expression of the liver-specific GHR mRNA variant homologous to ovine exon 1A was not observed in fetal liver examined between 11 and 30 wk of gestation but was detected in liver collected 1 wk after birth (17 , 33 ). There are also ontogenic changes in the other human GHR mRNA variants with loss of the exon 3 deleted isoforms from fetal tissues during the last part of gestation (22) . In pigs, perinatal up-regulation of hepatic GHR gene expression has been demonstrated in studies using probes specific to different regions of the porcine GHR gene (7 , 25 , 26 ). However, the onset and time course of the increment in hepatic GHR mRNA varied with the different probes (7 , 25 , 26 ). Developmental changes in GHR mRNA transcript composition may therefore also occur in the human and pig close to delivery when fetal cortisol levels are rising most rapidly in these species (27) . In the rat, the major perinatal increment in glucocorticoids occurs 3–4 wk after birth at the time of weaning (27) . This is the age at which, GHR1, the rat liver-specific GHR mRNA variant homologous to ovine exon 1A-containing transcript, is first detected in rat liver (12) . Glucocorticoids may therefore have an important developmental role in activating expression of the adult liver-specific GHR mRNA variant in many different species.
The mechanisms whereby cortisol induces expression of specific leader exons in the GHR gene are unknown. Leader exon 1A was switched on when fetal cortisol levels exceeded 35 ng/ml whereas expression of the other leader exons showed a more gradual ontogenic increase, which was closely related to fetal plasma cortisol levels over the entire range of concentrations observed in utero during late gestation. Hence, different molecular mechanisms may be used by cortisol to differentially up-regulate specific leader exons. At the transcriptional level, the actions of glucocorticoids are usually mediated by interactions between the hormone–nuclear receptor complex and promoter sequences, termed glucocorticoid response elements (GREs). Sequence analysis of the 5' flanking region of ovine GHR leader exon 1A has revealed two putative half site GREs within the immediate 669 bp region 5' to the start site for transcription of this exon (16) . By contrast, no glucocorticoid response element has been identified in the 1.3 kb region immediately upstream of the transcription initiation site for the ovine GHR leader exon 1B (15) . However, the upstream region of exon 1B appears to contain a putative half-site for binding the activated thyroid hormone receptor (15) , unlike the equivalent region of exon 1A (12) . Moreover, in several fetal tissues, the maturational effects of cortisol are known to be mediated by thyroid hormones 1-3) . Cortisol stimulates deiodination of thyroxine to triiodothyronine (T3) and leads to a concomitant rise in plasma T3 toward term (27) . Using probes to GHR coding exons, fetal thyroid hormone deficiency has been shown to prevent the normal up-regulation of hepatic GHR gene expression in fetal sheep and pigs close to term (25 , 34 ). Conversely, infusion of T3 into fetal sheep before term can prematurely elevate hepatic GHR gene expression (34) . Cortisol may therefore regulate hepatic GHR gene expression via both the 1A and 1B leader exons by acting directly on the exon 1A promotor and indirectly through T3 on exon 1B expression. In addition, other cortisol-dependent transcription factors or endocrine changes, such as the prepartum rise in plasma estradiol (27) , may be involved in regulating GHR gene expression in the fetal liver close to term. Estradiol is known to enhance hepatic GHR mRNA abundance in adult rats primarily through increased expression of the GHR1 variant, the rat homologue of ovine exon 1A (12 , 35 ).
Much less is known about the factors regulating GHR gene expression before the prepartum hormonal changes. Earlier in gestation, the hepatic GHR mRNA is derived entirely from leader exons other than 1A and probably originates largely from exon 1B (J. Li, R. S. Gilmour, and A. L. Fowden, unpublished observations). However, thyroid hormone deficiency in utero has little effect on hepatic GHR mRNA abundance in immature fetal sheep and pigs (25 , 34 ). In postnatal animals, hepatic GHR gene expression has been shown to be influenced by energy status, sex steroids and by IGF-I 35-37) . The relatively low levels of IGF-I in utero and the increased exposure to sex steroids during pregnancy may account, in part, for the basal levels of GHR gene expression observed in fetal liver before the prepartum cortisol surge (1 , 2 ).
The functional significance of alternate splicing in the 5' untranslated region of the GHR gene remains obscure. Differences in this region alter the structure of mRNA and modify its stability and translatability accordingly (38). Varying the transcript composition of mRNA may therefore provide a mechanism for altering the amount of protein produced. In sheep, an increase in GH binding to hepatic membranes, and hence in functional GHR protein, occurs during very late gestation (5) . This is followed by a marked increase in hepatic GH binding postnatally to reach high levels in the adult (5 , 18 ). These changes are correlated closely with the present findings for leader exon 1A expression: the first appearance of leader exon 1A-derived GHR mRNA occurs during late gestation, with a further marked up-regulation between birth and adult life. By contrast, the abundance of GHR mRNA derived from other leader exons, including 1B, is similar at birth to that in the adult. Together, these findings suggest that leader exon 1A-derived GHR may be the major contributor to the postnatal increment in hepatic GH binding and GH-dependent IGF-I production. In the sheep, fetus up-regulation of hepatic GHR gene expression close to term and in response to cortisol infusion is certainly associated with a rise in hepatic IGF-I mRNA abundance (24) .
The effects of cortisol on GHR gene expression are in keeping with its other known maturational effects in utero (2) . It has a key role in ensuring the successful transition from parenteral to enteral nutrition at birth and in adapting growth and metabolism to this new pattern of nutrition (27) . By activating the adult mode of GHR gene expression, the prepartum cortisol surge may initiate maturational changes in the somatotrophic axis that result in the onset of GH-dependent growth after birth. However, whereas preterm cortisol infusion replicated the normal prepartum rise in GHR gene expression from leader exons other than 1A, it did not appear to elevate exon 1A expression to the values observed in term fetuses with similar plasma cortisol concentrations. Furthermore, the abundance of GHR mRNA containing exon 1A in fetal liver at term was less than that in adult liver. Factors other than cortisol may, therefore, be involved in enhancing GHR exon 1A expression in ovine liver during the perinatal period. The identity of these factors and the molecular mechanisms by which cortisol acts on the various promotors of the ovine GHR gene remain to be determined.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Present address: Department of Molecular
Medicine, Auckland University School of Medicine, Auckland, New
Zealand.
3 Abbreviations: GH, growth hormone; GH, growth hormone
receptor; GRE, glucocorticoid response element; IGF, insuline-like
growth factor; PCR, polymerase chain reaction; T3,
triiodothyronine.
Received for publication May 28, 1998.
Revision received October 28, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
, upper panel) and total
hepatic GHR mRNA (lower panel) derived from either exon 1A (
) or
from the other leader exons (
) in sheep fetuses at different
gestational age. Values with different letters are significantly
different from each other (P<0.05).
+Significantly greater than values seen at gestational ages
130 days.