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Laboratory for Pregnancy and Newborn Research, Cornell University College of Veterinary Medicine, Ithaca, New York 14853
Address all correspondence and requests for reprints to: Dr. Peter W. Nathanielsz, Laboratory for Pregnancy and Newborn Research, Department of Biomedical Sciences, Cornell University College of Veterinary Medicine, Ithaca, New York 14853-6401. E-mail: pwn1{at}cornell.edu
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Abstract |
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At 109–113 d gestation, fetal sheep adrenals were removed (n =
8), or sham surgery was performed (n = 4). From d 6
postadrenalectomy, maternal and fetal plasma cortisol were determined
daily by RIA. From d 7 postadrenalectomy, cortisol (4 µg/min) was
continuously infused iv to four adrenalectomized fetuses. Endometrium,
myometrium, and placentome were collected from all three groups of ewes
(n = 4 for each group), and total RNA and proteins were extracted
from each intrauterine tissue and analyzed by Northern and Western for
prostaglandin G/H synthase 2 mRNA and protein. P45017
hydroxylase
mRNA was analyzed in the placentome by Northern blot. Data were
analyzed by ANOVA.
Plasma cortisol levels remained low in sham-operated and
adrenalectomized fetus, whereas during cortisol infusion to
adrenalectomized and cortisol-treated fetuses, plasma cortisol
increased to the late gestation level. After adrenalectomy,
prostaglandin G/H synthase 2 did not change in any tissue studied.
Fetal plasma cortisol replacement to late gestation levels increased
prostaglandin G/H synthase 2 to levels similar to term levels in all
three tissues. PGHS1 mRNA and protein did not change in any group
studied. There was a minimal increase in P45017 hydroxylase mRNA in
the placentome in the adrenalectomized and cortisol-treated group.
Cortisol- induced labor further increased P45017 hydroxylase
mRNA in the placentome compared with that in adrenalectomized and
cortisol-treated animals.
These data provide evidence for in vivo cortisol up-regulation of prostaglandin G/H synthase 2, but not PGHS1, in late gestation in the ovine placentome, myometrium, and endometrium. As stimulation of the estrogen biosynthetic pathway was minimal in the adrenalectomized and cortisol-treated group, these data provide support for the concept that cortisol has a direct effect on prostaglandin G/H synthase 2 expression in addition to its classical indirect pathway on prostaglandin G/H synthase 2 as a result of estrogen synthesis.
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Introduction |
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hydroxylase activity (5). Estrogen then recruits many
positive systems, including enhanced PG production in the intrauterine
tissues. This is the estrogen- regulated indirect pathway of
cortisol stimulation of PG production leading to premature labor in
pregnant sheep. Prostaglandin G/H synthase (PGHS) is the central enzyme controlling the PG biosynthetic pathway. Two forms of PGHS have been cloned and characterized; they are termed constitutive isozyme PGHS-1 and inducible isozyme PGHS-2 (6, 7, 8). PGHS-2 is considered responsible for stimulation of PG production in intrauterine tissues associated with the onset of labor (9, 10, 11). We conducted a range of studies on regulation of PGHS2 by estrogen in the nonpregnant sheep (12). Our approach was first to identify key regulators in the ovariectomized nonpregnant animal, a preparation that permits precise regulation of the steroid environment. We demonstrated that estradiol stimulated PGHS2 mRNA and protein expression in the endometrium and myometrium of the ovariectomized nonpregnant sheep. We therefore postulated that the positive effect of estradiol on uterine PGHS2 expression might extend into pregnancy. Indeed, our data obtained from pregnant sheep indicated that there were a temporal association between the peak of estradiol and the induction of PGHS2 in critical intrauterine tissues (13) in a tissue-specific manner. Placentome and endometrium developed gradually increasing abundance of PGHS2 message and protein during late gestation, whereas myometrial PGHS2 remains low throughout late gestation. PGHS2 in the myometrium, endometrium, and placenta are all up-regulated during labor.
However, several lines of evidence suggest that estrogen may not be the
sole regulator of PGHS2 in late gestation, and pathways such as the
direct cortisol stimulation of PGHS2 may exist. For example, Liggins
demonstrated that infusion of estrogen before 135 dGA into pregnant
ewes or near term into the fetuses could not induce the onset of
premature labor (1). We and others therefore proposed that
glucocorticoid may have a direct effect on intrauterine PGHS2
expression in addition to the indirect pathway through estrogen. Two
groups of investigators, Olson and co-workers (11, 14) and
Challis and co-workers (15), have shown in
vitro that glucocorticoid stimulates PG production directly in
cultured human fetal membranes. In sheep the abundance of placental
PGHS2 increases before placental P45017 hydroxylase increases
(16). These findings support an action of cortisol on PG
production by a direct pathway even before the indirect pathway via
estrogen is activated to prepare for the onset of labor. Furthermore,
evidence has also been presented for the existence of a
cortisol-dependent/estradiol-independent mechanism on PGHS2 induction
within trophoblast tissue leading to elevation of fetal plasma
PGE2 (17) in pregnant sheep.
Based on previous observations we hypothesize that cortisol may
directly (Fig. 1
) regulate PGHS2
expression in ovine placenta, endometrium, and myometrium in a
tissue-specific manner to initiate and maintain several processes
involved in labor in sheep in addition to the well established
estrogen-mediated indirect pathway. In the present study we examined
the effect of fetal cortisol on PGHS2 expression in the placentome,
endometrium, and myometrium by infusing cortisol into adrenalectomized
(ADX) fetuses at 125 dGA to reach the fetal plasma cortisol levels
present early in the late gestation rise, which are inadequate to
produce labor. Removal of endogenous fetal cortisol production by fetal
adrenalectomy (ADX) allowed identification of the specific function of
carefully controlled levels of cortisol in distinction to other
steroids of the fetal adrenal. We also examined placental
P450C17 mRNA expression to determine the
extent of involvement of the estrogen-mediated indirect pathway
induced by cortisol infusion on intrauterine PGHS2
expression.
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Materials and Methods |
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Placentome, myometrium, and endometrium were collected from all three groups: Sham, ADX, and ADX+F and from term control not in labor (TCNL; between 140–145 dGA; n = 4). Placentome was also collected from the cortisol-induced labor group. Tissues were flash-frozen in liquid nitrogen and stored at -80 C before total RNA or protein extraction.
RIA for maternal and fetal plasma cortisol
Plasma cortisol concentrations were measured using a
commercially available RIA kit (Diagnostic Products, Los
Angeles, CA) validated for measurements in sheep plasma
(18). The assay sensitivity, set at 90% bound/free, was
4.9 ng/ml (n = 27).
Total RNA preparation and Northern blot analysis
Total RNA from placentome, endometrium, and myometrium was
prepared as described previously (19). The RNA purity and
recovery of each tissue was determined by UV spectrophotometer (260 and
280 nM).
Samples of total RNA (40 µg/lane) were denatured in 17.4% (vol/vol) formaldehyde, 50% (vol/vol) formamide, 20 mM 3-(N-morpholino)propanesulfonic acid, 5 mM sodium acetate, and 1 mM EDTA, pH 7.0, for 5 min at 65 C and separated on a 1% (wt/vol) agarose/0.66 M formaldehyde gel. Ethidium bromide-stained ribosomal RNA (rRNA) bands were visualized (UV) to ensure that RNA degradation had not occurred and an equal amount of RNA was loaded into each lane. After electrophoresis, RNA was transferred to a nylon membrane (Gene Screen Plus, NEN Life Science Products, DuPont, Wilmington, DE) by capillary blotting for 24 h in 10 x SSC (1 x SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0). Prehybridization (>1 h) and hybridization (>18 h) were carried out at 42 C. The probe concentration was approximately 1 x 106 cpm/ml hybridization buffer [50% (vol/vol) deionized formamide, 50 mM sodium phosphate, 0.8 M NaCl, 2% (wt/vol) SDS, 100 µg salmon sperm DNA/ml, 20 µg transfer RNA/ml, and 1 x Denhardt’s (50x = 1% solution of BSA, Ficoll, and polyvinylpyrrolidone)]. Membranes were washed twice for 5 min each time in 2 x SSC and 0.1% SDS at 65 C temperature and twice for 15 min in 0.1 x SSC and 0.1% SDS at 65 C. The same membranes were reprobed for ß-actin or 18S.
cDNA probes
Recombinant sheep PGHS-2 cDNA and PGHS-1 cDNAs was purchased
from BIOMOL Research Laboratories, Inc. (Plymouth Meeting,
PA). P45017 hydroxylase probe was provided by Dr. Waterman (Dallas,
TX).
Solubilized cell membrane extraction and Western blot
analysis
Solubilized cell membrane extracts from myometrium, endometrium,
and placentome were prepared as described previously (10).
The protein concentration was determined by the method of Bradford
(Bio-Rad Laboratories, Inc.). The extracted protein
samples from different groups in each tissue were separated on the same
gel and stained with Coomassie blue to analyze IgG heavy and light
chains before performing Western blot analysis. We only used protein
samples that did not yield significant differences in these control
proteins. The solubilized proteins (100 µg/lane) were then separated
on 10% SDS-PAGE and electrophoretically transferred to a nylon
membrane (Immobilon, Millipore Corp., Bedford, MA), using
a transfer blot cell (Bio-Rad Laboratories, Inc.). Western
blot analysis was performed as described previously (10).
The protein bands were visualized using an enhanced chemiluminescence
Western blotting detection kit (ECL, Amersham Pharmacia Biotech, Arlington Heights, IL). The molecular sizes of the
proteins were determined by running standard molecular weight marker
proteins (Bio-Rad Laboratories, Inc.) in an adjacent lane.
Chemiluminescence signals were analyzed and quantified with the
scanner, and data were analyzed with a densitometry program, Scan
Analysis, and quantified against an arbitrary scale in the plot.
Antibodies for PGHS-1 and -2 used for Western blot
analysis
A rabbit polyclonal antibody for PGHS-2 raised against a
synthetic peptide corresponding to the C-terminal region of human
PGHS-2 (Oxford Biomedical Research, Inc., Oxford, MI) was used at a
1:1000 dilution and incubated at 4 C for 20 h. A mouse monoclonal
antibody for ovine PGHS-1 purchased from Oxford Biochemical Research,
Inc., was used at a 1:100 dilution and incubated at 4 C for 20 h.
PGHS-1 and PGHS2 antibodies have been characterized previously
(10, 12). Two different second antibodies, horseradish
peroxidase-conjugated sheep antimouse IgG (for PGHS-1) or horseradish
peroxidase-conjugated donkey antirabbit IgG (for PGHS-2), were
incubated with the membranes at room temperature for 1 h.
Statistical analysis
Comparison of three or more means was made using ANOVA, and
multiple post-hoc comparisons with Tukey’s method for 95%
confidence interval of pairwise differences. Statistical significance
was assumed at the 5% level. Data are presented throughout as the
mean ± SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). There was no significant difference
in maternal plasma cortisol concentration among the three groups,
except on d 1, when the ADX+F mothers showed significantly higher
cortisol concentrations than the Sham and ADX mothers (Fig. 3). No consistent interrelationship was
found between maternal and fetal plasma cortisol concentrations (Figs. 2 and 3).
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).
The trend
of changes in PGHS2 mRNA paralleled the changes at the protein level in
all three tissues examined (
Figs. 7–9).
ADX had no significant effect on PGHS2 mRNA. ADX with cortisol
replacement that produced plasma levels present early in the late
gestation fetal cortisol rise induced a significant rise in PGHS2 mRNA
in the endometrium (Fig. 7). The P values for the rise in
the myometrium and placentome were 0.06 and 0.08, respectively (Figs. 8 and 9). There was no difference in PGHS2 protein level between ADX+F
and TCNL groups in all three tissues studied (Fig. 10).
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P45017 hydroxylase
P45017 hydroxylase mRNA was undetectable in the placentome in
sham and ADX groups. However, there was a slight, but significant,
increase in P45017 hydroxylase mRNA in the placentome associated
with cortisol replacement to the fetus after adrenalectomy
(P < 0.05; Fig. 11).
This increase in P45017 hydroxylase mRNA in ADX fetuses induced by
fetal replacement of cortisol to levels present at the beginning of the
late gestation rise was much lower than the level of P45017
hydroxylase mRNA in the placentome during cortisol-induced premature
labor (Fig. 11).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In addition, it is important to note that the paradigm used in almost all previous experiments by different research groups including ours involved infusion of exogenous cortisol, whereas endogenous cortisol and, perhaps more importantly, other adrenal steroids continued to be produced by the fetus in situ. Furthermore, in these previous experiments glucocorticoid was generally used to induce the full range of changes associated with labor. Therefore, the changes in intrauterine PGHS2 observed were precipitated by the sum effect of hormones that continue to be produced by fetal hypothalamic-pituitary-adrenal activity in addition to effects of the exogenous infused glucocorticoid. By conducting fetal ADX in all animals investigated in the present studies, we removed endogenous fetal cortisol as well as other steroids of fetal adrenal origin with concurrent replacement of a known amount of cortisol to produce late gestation, but not delivery, levels. Removal of endogenous fetal cortisol production provided the ability to identify the specific function of carefully controlled levels of cortisol in distinction to other steroids of fetal adrenal. Although maternal adrenals were still intact, it should be noted that fetal plasma ACTH rises after fetal adrenalectomy, showing that maternal cortisol cannot maintain even basal fetal cortisol concentrations after fetal ADX at 120 dGA (18). Numerous in vivo data have shown that cortisol at the concentration used in this study does not cause the dramatic increase in estrogen and the fall in progesterone associated with the onset of labor (20, 21). We present the first in vivo evidence that these lower levels of fetal cortisol infusion, independent of any other steroids of fetal adrenal origin, resulted in increased PGHS2 expression in the endometrium, myometrium, and placentome before labor.
The present study extends our previous observations conducted using nonpregnant ovariectomized sheep and pregnant sheep to determine the regulation of uterine PGHS2 expression by estradiol or progesterone alone or in combination. In nonpregnant sheep we found that both estradiol and progesterone up-regulated PGHS2 expression in a uterine tissue-specific manner. During pregnancy, we determined tissue-specific and gestational age-dependent ontogenetic PGHS2 expression in myometrium, endometrium, and placenta during late gestation and spontaneous term labor in sheep. The abundance of PGHS2 in placentome and endometrium increased gradually during the last third of gestation well before the rise in plasma estrogen that occurs in the final hours of gestation (22). In contrast, myometrial PGHS2 remained low throughout late gestation. PGHS2 mRNA and protein increased in all three tissues only during spontaneous term labor (13). The gestation-dependent increases in placental and endometrial PGHS2 and fetal plasma PGE2 parallel the increase in fetal plasma cortisol concentration (13, 23) and occur at a time when the estrogen concentration is low and constant. The increase in PGHS2 in placenta, endometrium, and myometrium during spontaneous term labor was significantly greater than the increase that occurred during gestation and correlated to the time of a sharp and rapid surge of estradiol in maternal plasma associated with labor (22). We therefore hypothesized that the increases in placentome (and endometrial) PGHS2 in late gestation are under the control of the late gestation rise of fetal cortisol, whereas estradiol may control the labor-associated further rise in myometrial, endometrial, and placental PGHS2 levels.
Our present studies supported part of our hypothesis, namely that
circulating cortisol at late gestation levels induced placentome and
endometrial PGHS2. However, we also observed an increase in myometrial
PGHS2 despite the current view that myometrial PGHS2 is mainly under
estradiol control. We therefore determined the abundance of placental
P45017 mRNA expression, a key enzyme for placental estrogen
synthesis in this species, after fetal cortisol replacement. We
intentionally clamped fetal cortisol at levels observed very early in
the late gestation rise in fetal cortisol, several days before fetal
and maternal estrogen rises. These levels of fetal cortisol were
associated with a small increase in placentome P45017 mRNA that
approached one sixth of the dramatic increase in placental P45017
mRNA associated with cortisol-induced labor. Whether this minimal
increase in P45017 is the cause of the myometrial PGHS2 increase
mediated through increased estradiol production served as a rationale
for us to compare the PGHS2 level in ADX+F group with the PGHS2 levels
in three intrauterine tissues from TCNL ewes. In both situations, fetal
plasma cortisol circulates at similar levels. We did not see a
significant difference in PGHS2 in any of three tissues studied,
indicating that the increased estrogen synthesis induced by fetal
cortisol is not a sufficiently strong signal to stimulate PGHS2
expression above term levels before labor. Taken together our past and
present observations indicate that cortisol may have a direct effect on
intrauterine PGHS2 synthesis as well as the well established effect
mediated through estrogen.
Our data are consistent with the report by Whittle et al. (17) that both cortisol-dependent and estrogen-dependent PG production pathways exist in intrauterine tissues using an approach that differed from ours. These investigators determined changes in placental and uterine PGHS2 expression in a cortisol-induced labor model while at the same time they attenuated estradiol production by maternal infusion of the aromatase inhibitor 4-hydroxyandrostendione. Our study was designed to clamp fetal cortisol at the concentrations observed early in the late gestation rise to minimize estradiol production. In addition, the two studies complement each other, because they determined the effect of cortisol at different levels: levels present at labor in their study and levels seen early in the late gestation rise in our study of intrauterine PGHS2 expression. The cortisol levels used in both studies exist in the physiological situation, and it is important to note that in the physiological situation PGHS2 increases in placenta and endometrium in late gestation well before labor, with a further increase during labor (13, 24, 25). Furthermore, our study used fetal adrenalectomy to isolate the effect of cortisol from the effects of other fetal adrenal steroids. In contrast, the other study left the fetal hypothalamic-pituitary-adrenal axis intact, thereby maintaining the components of the fetal-placental unit. These two studies support each other by providing similar conclusions from different approaches toward understanding cortisol and estrogen regulation of PGHS2 associated with late gestation and labor in pregnant sheep.
It is of interest to note that fetal ADX did not significantly alter intrauterine PGHS2 expression in any of three tissues studied compared with that in the sham group. This indicates that basal PGHS2 at the stage of gestation is independent of cortisol production by fetal adrenal around 110–120 dGA.
Steroid-receptor complexes induce or repress the expression of genes by interacting with regulatory DNA sequences and transcription factor(s). The upstream promoter regions (up to 1.8 kb) of PGHS-2 genes from several species have been sequenced and do not appear to possess estradiol response elements (26, 27). However, the promoter for chicken PGHS2 contains an activator protein-1 site that may mediate the estrogenic effect on PGHS2 induction in intrauterine tissues. In contrast, a glucocorticoid response element has been identified in the promoter region of human PGHS2 (28, 29), providing a clear mechanism for the direct interaction of glucocorticoid with PGHS2 gene promoter. The ovine PGHS2 promoter has not been cloned, and potential regulators remain undetermined. High homology of the 5'-flanking region of PGHS-2 gene has been demonstrated in the human, murine, and rat PGHS-2 promoter, although dissimilarity was observed between the human and chicken PGHS-2 promoter sequences (27). Further study of the ovine PGHS-2 promoter region is necessary to obtain a better understanding of the regulatory mechanism of PGHS2 expression at critical time of pregnancy and during parturition.
The importance of PG in the preparation, activation, and stimulation of parturition has been recognized for over 30 yr. The limitations of our understanding of the regulation of PG production at a critical period of pregnancy and during parturition inhibit our management of preterm labor. The central location of PGHS in the PG biosynthetic pathway highlights its importance in the production of primary PGs. Our data again showed that PGHS2, not PGHS1, is under glucocorticoid control. Therefore, a better understanding of the mechanisms that control PGHS2 expression will greatly advance our knowledge of both normal and abnormal labor.
These data provide evidence for in vivo cortisol up-regulation of PGHS2, but not PGHS1, in late gestation in the ovine placentome, myometrium, and endometrium. As stimulation of the estrogen biosynthetic pathway was minimal in the ADX+F group, these data provide support for the concept that cortisol has a direct effect on PGHS2 expression in addition to its classical indirect pathway on PGHS2 as a result of estrogen synthesis.
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Acknowledgments |
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Footnotes |
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Abbreviations: ADX, Adrenalectomized, adrenalectomy; dGA, days gestation; F, cortisol infused; PGHS2, prostaglandin G/H synthase 2; rRNA, ribosomal RNA; Sham, sham adrenalectomized; TCNL, term control not in labor.
Received December 21, 2000.
Accepted for publication May 1, 2001.
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) of regulation PG production by fetal
cortisol. +, Positive stimulation.
