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Department of Obstetrics and Gynecology (A.-R.F.), Cornell University Medical College, New York, New York 10021; Département d’Ontogénie et Reproduction, Centre de Reserche du Centre Hospitalier de l’Université Laval, Centre de Recherche en Biologie de la Reproduction (P.D., M.A.F.), Ste Foy, Quebec, Canada; Institute for Hormone and Fertility Research (M.B.), Hamburg, Germany; Animal Science Department, University of Florida (M.J.F.), Gainesville, Florida 32611
Address all correspondence and requests for reprints to: Dr. A.-R. Fuchs, Department of Obstetrics and Gynecology, Cornell University Medical College, 515 East 71 Street, Room S-412, New York, New York 10021. E-mail: annariitta{at}aol.com
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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release in response to OT was
determined in 3-, 6-, and 9-month-old heifers (n = 4 each).
Myometrium, endometrium, and cervical mucosa were obtained from
3-week-old, 3-month-old, 6-month-old, and 9-month-old heifers and from
adult cows at estrus. Whole uterus and cervix were taken from third
trimester fetuses and at birth. [3H]OT binding and
specificity, localization of immunoreactive (ir) OTR, OTR messenger
RNA, and OT-induced release of PGF2 were determined. The
uterus from fetuses and the neonate expressed OTR messenger RNA and
bound [3H]OT. At 3 weeks of age, OTR concentrations per
mg protein were very low, but at 3 months of age they had increased
markedly in all three tissues. At 6 and 9 months of age, levels of OTR
had risen further and were similar to those in adult cows at estrus.
Prepubertal uterus also possessed separate vasopressin VP1 subtype
receptors. The ir-OTR was localized in luminal epithelial cells of
endometrium and cervical mucosa, most of which were ir positive,
whereas in myometrium, clusters of ir-OTR-positive cells were found
among large numbers of ir-OTR-negative cells. The PGF2
response to OT was insignificant in heifers of all age groups, in
contrast to that in cows at estrus. Endometrial cells from 4- to
5-month-old heifers did not respond to OT with PG release in the
absence or presence of added arachidonic acid. Tumor promoters,
lipopolysaccharide, and interleukin-2 also failed to elicit PG release
in vitro, although they induced PG release in similar
cell cultures from cyclic cows. In summary, uterine tissues of
prepubertal heifers have high levels of OTR, which appear to be
developmentally regulated. These receptors are not coupled to PG
synthase, or alternatively, the PG synthase gene is not expressed
before puberty, possibly because the tissues have had no previous
exposure to progesterone. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The influence of the specific endocrine conditions prevailing
before puberty on OTR concentrations in the reproductive tract has not
been established in any species. Indirect evidence from the poor
13,14-hydroxy-15-keto-PGF2 (PGFM) responses to OT
injections in prepubertal heifers (3) suggests that the endometrium may
not possess OTR at this stage. Fetal ovaries show significant activity
during the third trimester, with follicles at all stages between
primary to 5 mm (4). At birth, the ovaries contain small follicles,
3–5 mm in diameter (5), the number of which increases from birth
onward (6), with maximum numbers occurring around 4 months of age (7).
Waves of follicle growth to 8–9 mm size occur at regular intervals
from 8 months of age onward (8, 9), but no luteinized tissue has been
observed until shortly before the first ovulation (10). The prepubertal
ovarian activity results in low, but steady, plasma concentrations of
17ß-estradiol (E2) from 1 month of age until puberty
(1–3 pg/ml), whereas plasma progesterone concentrations are
undetectable (11). Elucidation of OTR ontogeny from birth to puberty
may provide new insights into the endocrine regulation of OTR in bovine
uterus. The purpose of this study was to investigate temporal aspects
of OTR expression and localization of the OTR protein in prepubertal
heifers from fetal age to adulthood. A second aim was to examine the
functional capacity of the putative OTR to stimulate
PGF2 release from the endometrium in vivo as
well as endometrial cell cultures in vitro.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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response to OT was determined in
other groups of 3-, 6-, and 9-month-old heifers and in a group of adult
cows at estrus of the same breed (n = 4 each). For in
vitro experiments, uteri from 4- to 5-month-old Limousin and
Charolais heifers were collected from a local abattoir, placed on ice,
and transported to the laboratory for further processing.
Blood sampling and OT injection
OT (50 IU) was injected into the jugular vein, and blood samples
were collected into heparinized tubes at 15-min intervals beginning 45
or 60 min before the injection of OT and for 90–120 min thereafter;
controls were given a saline injection iv. The blood samples were kept
on ice until plasma was separated and were kept frozen until
assayed.
PGFM assay
RIA for PGFM in unextracted bovine plasma has been described in
detail previously (12, 13). All samples were assayed in duplicate, and
the standard curves were constructed with buffered plasma from
ovariectomized cows instead of phosphosaline buffer. The cows had been
treated with a PG synthase inhibitor, Banamine (Schering-Plough Animal
Health Corp., Kenilworth, NJ; 20 mg, twice at a 12-h interval) before
collecting blood. The intra- and interassay variations during this
study were 3.5% and 6.7%, respectively; the detection limit varied
between 10–20 pg/ml.
OTR assay
Frozen tissues were pulverized and immediately homogenized in 10
mM Tris-HCl buffer containing the protease inhibitor
phenylmethylsulfonylfluoride. OTR concentrations were measured in a
crude microsomal fraction obtained by sequential centrifugation at
1,000 x g and 160,000 x g. The
protein concentration in assay tubes was 250-1000 µg/ml, depending on
the tissue examined; in this range, specific binding is linearly
related to protein concentration. Tritiated OT (New England Nuclear,
Boston, MA) was used as the labeled ligand in saturation assays, as
described previously in detail (14). Incubation was performed at 22 C
for 60 min, and separation was achieved by rapid filtration through
Whatman GF/F filters (Whatman, Clifton, NJ) with a cell harvester
(Braendel, Gaithersburg, MD). The assay buffer consisted of 50
mM Tris-maleate buffer, 5 mM MnCl2,
phenylmethylsulfonylfluoride (0.001%), and BSA (0.1%), pH 7.4. The
binding data were analyzed with a computerized curve-fitting program,
Ligand (Biosoft, Cambridge, UK), which calculates the parameters
Ka, Kd, binding capacity (Bmax),
and their statistical significance. Values were expressed as femtomoles
per mg microsomal protein. Unlabeled OT, arginine vasopressin (AVP),
and the VP1 antagonist called Manning Compound were purchased
from Bachem (Torrance, CA); isotocin, arginine vasotocin (AVT),
mesotocin, phenylalanyl2 ornithine8 vasopressin
(PAVP), and the specific antidiuretic agonist deamino-D-AVP
were purchased from Peninsula Laboratories (Belmont, CA); a specific OT
antagonist
[1-D(CH2)5,Tyr(Me)2,Thr4,Tyr-NH29]ornithine
AVT, and a specific linear vasopressin VP1-type antagonist were
gifts from Dr. M. Manning (Toledo, OH).
Endometrial cell cultures
Uteri from prepubertal (4–5 months old) heifers were collected
at the local abattoir within 15 min of exsanguination and kept on ice
until further processing at the laboratory. The successive steps of
endometrial cell explant culture were performed in sterile HBSS using a
modification of the method published previously (15). Briefly, each
uterus was freed from fat and annexa, and the two horns were cut open
to expose the luminal epithelium. The endometrium was then gently
scraped off with a sterile scalpel. The endometrial cell suspension, a
mixture of epithelial and stromal cells, was recovered by
centrifugation at 500 x g for 15 min. The cell pellets
were washed twice with HBSS. The final pellets were resuspended in RPMI
1640 medium (Flow Laboratories, McLean, VA) supplemented with 50
µg/ml gentamicin (Sigma Chemical Co., St. Louis, MO). Cells were then
plated in 24-well culture plates (Becton Dickinson, Lincoln Park,
NJ).
One hour after plating, the medium was replaced with 1 ml fresh medium containing OT (10-11-10-5 M) alone or combined with arachidonic acid (AA; 0.34 mM). Samples were incubated at 37 C in a humidified atmosphere of 95% air-5% CO2 for 24 h in triplicate. The experiments were repeated four times with tissues from different heifers. In another experiment, various other compounds were added instead of OT with the fresh RPMI 1640 medium 1 h after plating. The compounds were 4ß-phorbol 12-myristyl 13-acetate (PMA; 10-10–10-8 M), lipopolysaccharide (LPS; 10 µg/ml), and interleukin-2 (IL-2; 1 and 10 ng/ml) alone or with AA (0.34 mM). At the end of the incubation, the culture medium was recovered for PG measurement from all wells and stored at -20 C until further processing. The plates were rinsed with ethanol, and DNA content was determined for each well by 3,5-diaminobenzoic acid fluorescence according to the method of Fiszer-Szafarz et al. (16) to estimate cell number for each well and standardize the results. The chemicals were obtained from Sigma.
PGF2|ga assays
Measurement of PGF2 was performed with an
in-house enzyme immunoassay technique that employs
acetylcholinesterase-linked PG tracers (Cayman Chemical Co., Ann Arbor,
MI) and fully characterized antibody developed in sheep according to
the method described in detail previously (17). The inter- and
intraassay coefficients of variations were 16% and 10%, respectively
(n = 12).
Immunohistochemical localization of bovine OTR
Immunoreactive (ir-) OTR protein was determined in frozen
sections (7 µm) of endometrium, myometrium, and cervical mucosa of
two heifers in each group by methods described previously in detail
(18). Briefly, tissues were mounted on gelatin-coated slides and
stained by the immunoalkaline phosphatase technique (APAAP complex,
Dianova, Hamburg, Germany) using a monoclonal antibody against human
OTR (provided by Dr. T. Kimura, Osaka University. Medical School,
Osaka, Japan) in dilutions of 1:500, 1:1000, and 1:2000. The antibody
was raised in mice against amino acids 20–40 in the N-terminal
extracellular region of the human OTR (19). This antibody was
previously found to cross-react extensively with bovine OTR (20, 21).
Control sections were treated with an equivalent amount of pure mouse
IgM instead of the antibody. Bright green with no counterstain was used
for visualization of the ir-OTR protein.
Ribonuclease (RNase) protection assay (RPA) for bovine OTR
transcripts
OTR messenger RNA (mRNA) was determined in selected samples by
RPA using kits from Ambion (Austin, TX) according to the
manufacturer’s instructions. The details of preparation of the
complementary RNA (cRNA) probe from PCR-amplified bovine OTR
complementary DNA cloned into the plasmid vector pCRII (Invitrogen,
Carlsbad, CA) have been described in detail previously (21). Ten
micrograms of total RNA from the tissue to be examined were hybridized
with the OTR-specific cRNA probe and, as a control for equal loading on
the gel, a bovine glyceraldehyde phosphate dehydgrogenase
(GAPDH)-specific cRNA probe in the same reaction vial. Because of the
differences in size of the protected fragments of the two cRNAs, they
could be readily be separated on a polyacrylamide gel.
Statistical analysis
The receptor concentrations in each tissue at the different ages
were compared with one-way ANOVA, followed by Fisher’s least
significant difference procedure. PGFM responses to the OT injections
were quantified as the area under curve according to Simpson’s Rule
(22), and differences among groups were assessed with one-way ANOVA and
Scheffe’s multiple contrasts. The data obtained from the in
vitro experiments were subjected to logarithmic transformation and
least squares ANOVA using the SuperAnova software package (Abacus
Concepts, Berkeley, CA). Sources of variation included treatment, dose,
time, and their interactions. Orthogonal contrasts were used to compare
treatments. The results shown are antilog values of the least square
means expressed as a percentage of the control value (PG values in
cells without addition of OT or other experimental agents).
P 
0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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At this age, OTR concentrations, with the exception of cervical mucosa,
had reached similar levels as those in adult heifers at estrus. The
results are summarized in Fig. 1
. At
birth, the concentration of uterine OTR was greater than that in third
trimester fetuses. OTR density in cervical tissue was higher than that
in uterus at both fetal stages. Postnatally, OTR concentrations per mg
protein decreased. At 3 weeks of age, receptor density was similar to
that in third trimester fetuses, but at 3 months of age, OTR density
had increased significantly in all three tissues (P <
0.05 for myometrium and cervix; P < 0.001 for
endometrium). A 10-fold increase in endometrial OTR concentrations and
2-fold increases in myometrium and cervical mucosa were observed in
this age group. At 6 months of age, OTR density in myometrium and
cervical mucosa had tripled from levels at 3 months, and endometrial
receptor density had doubled (P < 0.002 for
myometrium; P < 0.05 for endometrium and cervical
mucosa). At 9 months of age, no further increase in OTR was observed.
Endometrial concentrations of OTR in cyclic heifers at estrus were
similar to those in 6- and 9-month-old heifers, whereas myometrial OTR
density was lower (P < 0.05) and cervical mucosal
OTR density was higher than those in 6- and 9-month-old heifers
(P < 0.002).
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(Kd = 1/Ka).
Linear regression for individual endometrial Ka values in
relation to age was significant (r = -0.535; P <
0.05). The Ka values for myometrial OTR showed a similar
tendency, but regression was not significant (P <
0.20). The mean values for Kd in endometrium, myometrium,
and cervical mucosa were similar: 0.81, 0.86, and 0.88 nmol/liter,
respectively.
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. The data
for AVP and several other agonists indicated binding to a heterologous
population of sites. The Bmax values for the two sites
suggested that there was a high affinity binding site for AVP of
smaller capacity than the OT site. The oxytocic analogs bound to the
AVP site with lower affinity than to the OT site, whereas the
vasopressor analog, PAVP, bound with higher affinity to the AVP site
than to the OT site. Using the highly specific vasopressin VP1 receptor
antagonist, [125I]linear vasopressin VP1-type antagonist,
the presence of a high affinity vasopressor receptor was verified, the
Bmax of which was substantially smaller than that of OT
(480 fmol/mg protein). These experiments suggest that the endometrium
of prepubertal heifers possesses separate receptors for OT and AVP, but
the receptors discriminate poorly between OT and AVP.
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and 3; the corresponding controls treated
with pure mouse IgM instead of antibody are also shown. At all ages,
ir-OTR was localized primarily in the luminal epithelial cells of
endometrium and cervical mucosa, where most cells appeared to be
stained for ir-OTR; glandular epithelial cells were less extensively
ir-OTR positive than luminal cells. By contrast, only a fraction of
myometrial cells were immunopositive for OTR, clusters of cells stained
for ir-OTR were scattered among many immunonegative cells.
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) and cervix (Fig. 2C) of late
trimester fetuses showed clear signals for ir-OTR in the luminal
epithelium of endometrium and cervical mucosa (corresponding controls
in Fig. 2, B and D). No immunoreactivity was observed in the myometrial
portion of the uterus or cervix. During bovine embryogenesis and fetal
uterine development, the cervix attains adult form earlier than the
endometrium and myometrium (4), which may explain the finding of strong
OTR expression in cervical epithelium, but no signals in myometrial
portion of the uterus.
Figure 2E shows ir-OTR in endometrial tissue from a 3-week-old heifer
with corresponding control in Fig. 2F. Surprisingly, luminal epithelial
cells of both endometrium and cervical mucosa (not shown) were strongly
stained for ir-OTR; staining intensity equaled that in older age
groups, although the density of binding sites was much lower.
Endometrial and myometrial tissues from a 3-month-old heifer are shown
in Fig. 3, A and C, with controls in Fig. 3, B and D. Increased
glandularity of the endometrium compared to that in 3-week-old heifers
is evident. The ir-OTR density was greatest in the luminal epithelium,
but ir-OTR staining was also detected in the epithelium of many, but
not all, glands of the 3-month-old animals. Only a minority of
myometrial cells was stained for ir-OTR (Fig. 3C); these cells occurred
in clusters among large numbers of immunonegative cells. The ir-OTR in
cervix had similar distribution as those in the fetal and 3-week-old
groups, with mucosal epithelial cells stained for ir-OTR but no stain
in the cervical wall (not shown).
Localization of ir-OTR in endometrium and myometrium of a 6-month-old
heifer is shown in Fig. 3, E and G; cervical mucosa of the same heifer
is shown in Fig. 2G, and corresponding controls are presented in Fig. 3, F and H, and Fig. 2H. Luminal epithelium and epithelial cells of all
deeper lying glands (Fig. 3E) showed strong signals for ir-OTR; many
subepithelial stromal cells also appeared to be stained. Myometrial
cells were now organized in fibrils and showed much stronger signals
for ir-OTR than they had at 3 months of age (Fig. 3G). Luminal
epithelial cells of cervical mucosa from 6-month-old heifers were
uniformly stained for ir-OTR (2G). Diffuse staining was also observed
in some submucosal areas.
Endometrium, myometrium, and cervical mucosa of 9-month-old heifers showed a similar distribution of ir-OTR as 6-month-old heifers (not shown). In adult heifers, endometrium, myometrium, and cervical mucosa had a similar distribution of ir-OTR as in the 9-month-old heifers (not shown) (20). Luminal epithelial cells were uniformly stained, and epithelial cells of many, but not all, deeper lying glands showed strong signals for ir-OTR, whereas no signals were seen in other cells. Myometrial cells of adult heifers at estrus were not uniformly stained for ir-OTR; groups of immunopositive cells were surrounded by numerous immunonegative cells as in the younger heifers.
OT-induced release of PGFM in vivo
Peripheral plasma PGFM concentrations in 3-, 6-, and
9-month-old prepubertal heifers and in cyclic heifers at estrus are
shown in Fig. 4. Basal levels before
injection of 50 IU OT were significantly lower in prepubertal heifers
than in cyclic cows (P < 0.001). Only one of the
3- and 6-month-old heifers each and three of the 9-month-old heifers
had detectable PGFM levels before the injection of OT (detection limit,
17 pg/ml), whereas basal levels in four estrous cows averaged 48.7
± 3.7 pg/ml (n = 28). Assay sensitivity was 7 pg/ml when plasma
samples from the 3-month-old heifers were analyzed; in these, the mean
basal plasma PGFM concentration was 15.9 ± 1.1 pg/ml. A brief
increase to 32 ± 5 pg/ml in plasma PGFM was observed 15 min after
OT injection in the 3-month-old group, but no significant increase
in the 6-month-old group was observed, and in the 9-month-old group, a
transient, small increase was seen. By contrast, OT elicited a
substantial, long lasting increase in plasma PGFM in estrous heifers.
The responses, quantified as the area under the curve, are shown in
Table 3. The differences among groups
were highly significant (by ANOVA: F = 10.00; P <
0.01). The responses in adult cows were much greater than those in the
prepubertal 3-, 6-, and 9-month-old heifers.
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release in prepubertal
heifers was caused by either a defect in the signal transduction
pathway or the absence of PG endoperoxide synthase or phospholipase
A2 from the tissues. To examine these alternatives, we
measured the effect of OT on PGF2 release in primary
cultures of endometrial cells obtained from four 4- to 5-month-old
heifers. The experiments were performed with and without added free AA.
We also measured PG output in response to PMA, LPS, and IL-2 in
endometrial cells from another group of 4- to 5-yr-old calves. PMA
bypasses the receptor/signal transduction step and stimulates protein
kinase C directly. PMA has been shown to increase PG production by
induction of PGH synthase-2 (PGSH-2) gene expression (also known as COX
II) (23). LPS and IL-2 also induce the expression of the PGSH-2 gene
and thereby increase PG output from cells (24, 25).
OT in concentrations (10-7 M) that elicit
PGF2 release in primary epithelial cell cultures from
cyclic cows (26) did not have any effect on the output of
PGF2 from cell cultures derived from prepubertal heifers
or have an effect in any concentration between
10-11–10-5 M (Table 4). In the presence of 0.34
mM AA, the endometrial cell cultures from prepubertal
heifers produced about 3 times more PGF2 than without
added AA, but the addition of OT to cell cultures with AA did not
increase PGF2 output further, in contrast to the effect
of OT in cell cultures derived from cyclic cows (17). Additions of PMA,
LPS, or IL-2 were likewise without effect, or increased PG output only
marginally (Table 5). These compounds
also failed to increase PGF2 production in the presence
of added AA. The effects of OT, PMA, LPS, and IL-2 on PGE2
production in the cell cultures were similar to those shown of
PGF2 (values not shown).
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). Distinct, but weak, signals of the
right length (protected fragment, 302 bases) with GAPDH as the
control (protected fragment, 198 bases) were detected after
separation on polyacrylamide gels and autoradiographic exposure for
48 h. Signals for OTR mRNA in endometrium and myometrium of cyclic
heifers at estrus, shown for comparison, were strong.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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OTR concentrations in all three tissues increased until 6 months of age, when receptor concentrations in endometrium equaled and those in myometrium exceeded those in cyclic cows at estrus. On the other hand, OTR density in cervical mucosa did not reach adult estrous levels before puberty. Tissue localization of ir-OTR was the same at all developmental stages, with luminal epithelium of the endometrium and cervical mucosa showing the greatest intensity of staining for ir-OTR. Distribution of ir-OTR in the epithelial cells of deeper glands was less uniform, and many glands showed no signals for ir-OTR. Myometrial cells in both prepubertal and cyclic cows expressed OTR only patchwise. This is in striking contrast to late pregnant bovine myometrium, in which practically all cells exhibited ir-OTR (Fuchs, A.-R., and M. Balvers, unpublished observations).
The changes in endometrial OTR concentrations paralleled increases in endometrial glandularity (27) and luminal epithelial cell height (5). Myometrial cells in the newborn calf are relatively undifferentiated and are not arranged in fibrils and bundles until a later age, when these developmental changes are associated with increased OTR concentrations. It has been reported that endometrial differentiation and development continue normally after ovariectomy at birth (28, 29), suggesting that these morphogenetic events are not dependent on ovarian hormones. The expression of OTR in the reproductive tract of prepubertal heifers is, therefore, likely to be developmentally regulated and steroid independent.
Whether the steady, but low, circulating levels of E2 (2–3
pg/ml) are directly responsible for the progressive increase in uterine
OT receptor concentrations from the early neonatal period until 6
months of age or are indirectly responsible through induction of
uterine growth and glandular development cannot be decided on the basis
of the present results. Considering that plasma E2 levels
in the periestrous period are significantly higher than those in
prepubertal heifers (
20 pg/ml), but uterine OTR concentrations in
endometrium and myometrium of estrous heifers are not higher than those
in 9-month-old prepubertal heifers, estrogens may not directly
stimulate OTR gene expression, but play an indirect, permissive role in
the formation of uterine OTR. Evidence for this view is provided by the
observation that ovariectomy of 9-month-old heifers did not result in
significant lowering of endometrial and myometrial OTR concentrations
(Fuchs, A.-R., W. Rust, M. Drost, S.-M. Chang, and M. J. Fields,
unpublished observations). OTR in cervical mucosa were, however,
markedly reduced, as was the protein concentration. We attribute the
reduction in OTR in cervical luminal epithelial cell to physiological
changes after estrogen withdrawal, as the luminal epithelial cells are
among the most estrogen-sensitive cells of the reproductive tract. Our
results are in accordance with the lack of estrogen response elements
(ERE) in the bovine OTR gene promoter (30). Therefore, the effect of
estrogen on OTR density in bovine reproductive tract depends on
estrogen-induced mediators and not on direct activation of the OTR
transcription system by E2. Similar considerations apply to
the human OTR gene. The promoter of the human OTR gene does not have an
ERE sequence within 7 kb upstream, and none of the ERE half-sites
responded with increased activity to E2 when cotransfected
with estrogen receptor (31). Conflicting results were recently reported
for the rat OTR gene (32), in which previously no ERE sequences had
been detected (33). The authors of the report described finding a
"missing" sequence in the promoter region of rat OTR gene in which
an ERE was present. In transfection experiments with the novel,
ERE-containing rat OTR gene promoter, the researchers observed only a
small response to E2, amounting to a 50% increase in
reporter activity from control levels in 24 h (32). However, OTR
protein in rat myometrium in vivo is unequivocally increased
after treatment with E2 (34, 35), which is not the case in
the bovine (Fuchs, A.-R., W. Rust, M. Drost, S.-M. Chang, and M. J.
Fields, unpublished observations) or human (36).
The decrease in receptor affinity during prepubertal development could indicate that slight conformational changes in the ligand-binding domain of the receptor protein occur during development. Another explanation would involve developmental changes in the lipid composition of the plasma membranes in which the receptor protein is embedded, a factor shown to influence ligand binding affinity of OTR (37).
Our study indicates that endometrial OTR are not functionally coupled to PG synthase in prepubertal heifers, or alternatively, PG synthase is not expressed in the uterine tissues of prepubertal heifers. The in vitro experiments indicated that endometrial cells derived from prepubertal heifers are capable of PG synthesis, albeit at much lower rates than cells from adult bovine endometrium. The paucity of phospholipase A2 was not responsible for the failure of OT to induce PG release, because addition of AA did not reverse this failure. Other agents known to elicit PG release by induction of PGSH-2 (23, 24, 25) also failed to elicit PG release from endometrial cells from prepubertal heifers in the presence and absence of added AA; therefore, the alternative explanation, lack of expression of the PGHS-2 gene, seems closer to the truth. Indeed, in cultures of endometrial cells from cyclic cows, OT stimulated PGHS-2, but not PGHS-1, mRNA expression (38), and in pregnant cows, OT injections caused within 2 h a significant increase in endometrial PGHS-2 mRNA expression (Fuchs, A.-R., and W. Rust, unpublished observations). We, therefore, infer that OT mediates PG release from endometrial epithelial cells through induction of PGSH-2 transcription. The gene for the constitutively expressed PGSH-1 is probably expressed in prepubertal bovine uterus at low concentrations, but the gene for the inducible PGSH-2 is not. According to Eggleston et al. (39), progesterone is required for the expression of PG synthase in ovine uterine tissues, an observation compatible with our findings in prepubertal heifers. The existence of two distinct PG synthases was not established at the time those experiments were conducted, but Eggleston et al. were probably measuring PGHS-2 and not PGHS-1 gene expression. Various tissues of the reproductive tract have since been shown to express PGHS-2 upon appropriate stimulation (40).
In summary, the OTR gene is expressed in the fetal bovine uterus in the
late third trimester if not earlier. Postnatally, the expression of OTR
increases rapidly, and high levels of receptor protein are maintained
in the luminal epithelium of endometrium and cervical mucosa throughout
puberty. The changes in OTR concentrations with age are probably
developmentally determined rather than induced by circulating
E2. The low E2 levels present in heifers before
puberty (2 pg/ml) probably enhance OTR expression indirectly by
promoting growth and differentiation of the uterus and cervix. OT does
not elicit PG release from the endometrium of prepubertal heifers,
apparently because the uterus does not express the inducible PGHS-2
gene before puberty.
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Acknowledgments |
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Footnotes |
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Received October 28, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 V. Terzidou, S. R. Sooranna, L. U. Kim, S. Thornton, P. R. Bennett, and M. R. Johnson Mechanical Stretch Up-Regulates the Human Oxytocin Receptor in Primary Human Uterine Myocytes J. Clin. Endocrinol. Metab., January 1, 2005; 90(1): 237 - 246. [Abstract] [Full Text] [PDF]
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 A. K. Goff Steroid Hormone Modulation of Prostaglandin Secretion in the Ruminant Endometrium During the Estrous Cycle Biol Reprod, July 1, 2004; 71(1): 11 - 16. [Abstract] [Full Text] [PDF]
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J. Parent, P. Chapdelaine, J. Sirois, and M. A. Fortier Expression of Microsomal Prostaglandin E Synthase in Bovine Endometrium: Coexpression with Cyclooxygenase Type 2 and Regulation by Interferon-{tau} Endocrinology, August 1, 2002; 143(8): 2936 - 2943. [Abstract] [Full Text] [PDF]
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S. L. Rigby, R. Barhoumi, R. C. Burghardt, P. Colleran, J. A. Thompson, D. D. Varner, T. L. Blanchard, S. P. Brinsko, T. Taylor, M. K. Wilkerson, et al. Mares with Delayed Uterine Clearance Have an Intrinsic Defect in Myometrial Function Biol Reprod, September 1, 2001; 65(3): 740 - 747. [Abstract] [Full Text] [PDF]
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