From the Unité de Recherche en Ontogénie et Reproduction et Département d'Obstétrique et Gynécologie, Centre Hospitalier Universitaire de Québec, Université Laval, Sainte-Foy, Québec G1V 4G2, Canada
Received for publication, August 14, 2002, and in revised form, December 18, 2002
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ABSTRACT |
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Prostaglandins are important regulators of
reproductive function. In particular, prostaglandin F2 Prostaglandins are local mediators acting through paracrine or
autocrine mechanisms. Prostaglandins are produced from arachidonic acid liberated from phospholipid stores through the action of phospholipases. Arachidonic acid is then converted into prostaglandin H2
(PGH2),1 the
common precursor of all prostaglandins, through the cyclooxygenase and
peroxydase activities of prostaglandin H synthase (PGHS). There are two
PGHS: PGHS1 and PGHS2. These enzymes (also known as Cox-1 and
Cox-2), which have been identified some 10 years ago, are still
extensively studied. Because PGH2 is the common precursor
of all subtypes of prostaglandins and because these prostaglandin
isotypes cause different and even opposing actions, the pathways
leading to their individual formation need to be identified.
Prostaglandin F2 PGF2 Until now, a total of six PGFS have been identified. The first three
were isolated in the cow: lung type prostaglandin F synthase (PGFS1)
(12), lung type PGFS found in liver (PGFS2) (13), and liver type PGFS,
also called dihydrodiol dehydrogenase 3 (DDBX) (14, 15). The three
other PGFS were respectively isolated from humans (16), sheep (17), and
Trypanosoma brucei (18). As a group, these enzymes belong to
the aldoketoreductase family (19). The enzyme from
Trypanosoma belongs to the AKR5A subfamily, whereas the
others belong to the AKR1C family, which is also primarily associated
with hydroxysteroid dehydrogenases. With the exception of the
Trypanosoma enzyme, these enzymes also possess a
PGD2 11-ketoreductase activity, thus giving them the
ability to convert PGD2 into 9 9-Ketoprostaglandin reductase activity has been detected in the
reproductive system of several species. In the rabbit, a 9K-PGR that
possesses 20 Studies on the regulation of PGFS1 have already been performed on
cultured bovine endometrial cells (11, 25, 26). Treatments with
hormones, oxytocin, and interferon The objective of the present work was to identify the metabolic
pathways and the enzymes involved in the formation of
PGF2 Materials--
Culture plates and Luria broth media were
purchased from Becton Dickinson (Lincoln Park, NJ). RPMI 1640 was
obtained from ICN Biomedicals (Aurora, OH). TRIZOL, fetal bovine serum,
Moloney murine leukemia virus-RT, restriction enzymes, and
buffer were purchased from Invitrogen. Oxytocin, hematoxylin (Mayer),
3- amino-9-ethyl-carbazol tablets, NADPH, and phenanthrenequinone
were obtained from Sigma. Recombinant ovine interferon Endometrial Tissue Collection and Epithelial Cell
Culture--
Bovine endometrial tissues were obtained by scraping the
interior of uterine horns obtained from a local abattoir within 2 h of slaughter. The days of the estrous cycle were determined by
macroscopic examination of both ovaries and uterus as described recently (28). Primary cultures of bovine endometrial epithelial and
stromal cells were performed as described previously (10, 29). Oxytocin
and interferon HPLC Analysis of Prostaglandin Metabolism--
Bovine
endometrial epithelial cells were grown to confluence, and the medium
was changed for serum-free medium to which was added oxytocin to
stimulate prostaglandin production. 1 µCi of radioactive precursor
([3H]arachidonic acid, [3H]PGE2
or [3H]PGD2) was used as substrate for
prostaglandin synthesis. After 24 h, prostaglandins present in the
supernatant were extracted with ethyl acetate (10), applied on a C18
column, and eluted with an acetonitrile:water:acetic acid (45:55:0.05)
solution. Peak assignment was performed with tritiated prostaglandin standards.
Complete Sequence Determination of PGFSL1 and PGFSL2--
For
PGFSL1, 5' and 3' RACE were performed based on the published partial
sequence (11). Total RNA from oxytocin-treated (6 h) bovine endometrial
epithelial cells was extracted using TRIZOL. cDNA first strand was
synthesized with the Ready-to-Go T-primed first strand kit. This
cDNA possesses a tag on its poly(A) end that can be used for 3'
RACE. 3' RACE was accomplished by PCR (30 cycles, denaturation at
94 °C for 30 s, annealing at 55 °C for 30 s and
transcription at 72 °C for 1 min) using oligonucleotides RACE3prime
and 35 (Table I). A ~700-bp fragment
was obtained and cloned in pCR2.1 using the TOPO TA-Cloning pCR2.1 kit.
Sequence analysis (using the T7 DNA polymerase sequencing kit) of
several clones revealed a new sequence highly homologous to the known bovine PGFS but different from the previously identified fragment of
PGFSL1. This new putative PGFS was named "PGFSL2." A second 3' RACE
assay for PGFSL1 was performed with liver cDNA. First, an
asymmetric PCR (30 cycles, annealing at 55 °C) was done using oligonucleotide 35 followed by PCR (30 cycles, annealing at 55 °C)
with oligonucleotides RACE3prime and 9KBOVs. The resulting 600-bp
fragment was cloned and sequenced as previously described. This
fragment corresponds to the 3' end of PGFSL1. 5' RACE of PGFSL2 was
performed by PCR (30 cycles, annealing at 55 °C) on oxytocin-treated
(6 h) bovine endometrial epithelial cell cDNA with oligonucleotides
36 and F1C7s (F1C11s and FDDBXs did not work). 5' RACE of PGFSL1 was
performed by PCR (30 cycles, annealing at 55 °C) on liver cDNA
with the oligonucleotides 9KBOVas and FDDBXs (F1C11s worked but not
F1C7).
Northern Blot Analysis--
The full-length coding sequences of
lung and liver type PGFS, DDBX, PGFSL1, PGFSL2, and AKR1B5 were
obtained by PCR (30 cycles, annealing at 50 °C) with the appropriate
oligonucleotides (Table I; oligonucleotides with names beginning with
F) using bovine lung or liver cDNA. The resulting fragments were
cloned with TOPO TA-Cloning pCR 2.1 kit and sequenced as above. A
BstXI digestion of those plasmids generated the probe that
was labeled with [ RT-PCR Analysis--
Total RNA from specified sources was
extracted using TRIZOL according to the manufacturer's instructions.
Reverse transcription was performed on 1 µg of RNA with Moloney
murine leukemia virus-RT as described by the supplier's protocol. PCR
amplification of a given gene was performed with its corresponding
oligonucleotides (Table I; oligonucleotides with names beginning with
F) for 30 cycles with an annealing temperature of 50 °C yielding a
fragment of ~1000 bp, except for PGFSL2 where primers FF3s and 36 yielded a fragment of 760 bp. PCR amplification of RNase Protection Assay--
Antisense riboprobes for AKR1B5 and
Enzymatic Activity--
The full-length coding sequence of each
gene was inserted in the NdeI restriction site of pET16B,
and the His tag proteins were produced and purified as described by the
manufacturer. Enzymatic activity was measured by monitoring NADPH
degradation at 340 nm. The assays were performed in 1 ml of 50 mM Tris-HCl, pH 7.5, 100 µM NADPH with
10-100 µg of enzyme and variable concentrations of the tested
compounds (17-hydroxyprogesterone, 17-hydroxypregnenolone, phenanthrenequinone, and PGH2). Phenanthrenequinone was
used as a universal AKR1C substrate to confirm the functionality of the enzymes. The production of PGF2 Western Blot and Immunohistochemistry Assays--
Rabbit
immunizations were performed using 4 × 250 µg of purified
recombinant AKR1B5 protein. Soluble endometrial proteins were obtained
by homogenization of endometrial tissue in 20 mM Tris-HCl,
1 mM phenylmethylsulfonyl fluoride, followed by
centrifugation at 13,000 × g for 10 min. Proteins in
the supernatant were then quantified as described previously (31).
Western blot was performed using 20 µg of protein/lane on a 10%
polyacrylamide gel. The proteins were transferred onto a nitrocellulose
membrane. A 1:5000 dilution of AKR1B5 antiserum and 1:10,000 dilution
of goat anti-rabbit secondary antibody were used. The membranes were
washed between antisera incubation with phosphate-buffered saline
containing 0.05% Tween. Revelation was performed using the Renaissance
kit, following the manufacturer's instructions. Immunohistochemical staining was performed as described in the instruction manual of the
Vectastain Kit (Paraffin section) using 1:750 dilution of AKR1B5
antiserum and hematoxylin as counterstain.
PGF2 Sequence of PGFSL1 and PGFSL2--
The complete nucleotide coding
sequences of PGFSL1 and PGFSL2 were obtained as
described under "Experimental Procedures" (data not shown;
GenBankTM accession numbers AY135400 and AY135401).
PGFSL1 and PGFSL2 are highly related to the three
known bovine PGFS. The nucleotide coding sequence of
PGFSL1 is 88% identical to those of PGFS1 and PGFS2, 90% identical to DDBX, and 93% identical
to PGFSL2. PGFSL2 is 89% identical to PGFS
1 and 2 and 90% identical to DDBX. At the
amino acid level, PGFSL1 is predicted to be 82% identical to PGFS1,
81% identical to PGFS2, 84% identical to DDBX, and 90% identical to
PGFSL2. PGFSL2 should be 83% identical to PGFS1 and DDBX and 82%
identical to PGFS2.
Northern Blot Analysis--
Northern blot analysis was performed
to determine the expression of PGFS1, PGFS2,
DDBX, PGFSL1, and PGFSL2 in the
endometrium and in primary cultures (Fig.
3). Extracts from lung and liver were
used as positive controls. Lanes 1-6 correspond to
different periods of the estrous cycle. A signal is present for all
probes but only at the beginning of the cycle. A strong signal was
observed for all probes in lung and liver controls, but no signal was
observed for cultured endometrial cells (either epithelial or
stromal).
RT-PCR Analysis--
RT-PCR analysis was performed in the same
tissues described above to increase the sensitivity of gene expression
detection by comparison with Northern blots. In Fig.
4, we show the expression of the three
known PGFS in the bovine endometrium throughout the cycle
(panel i) and in cultured epithelial cells (panel
ii). Apart from the lung controls, no signal was visible for
PGFS1 and 2. A weak signal was found for
DDBX at the beginning of the cycle and in control
(untreated) epithelial cells, but this weak signal disappeared in all
other conditions. The Identification of an Alternate PGFS--
These results show that
of a massive production of PGF2 Enzymatic Activity--
PGFS1, PGFS2, DDBX, PGFSL1, PGFSL2, and
AKR1B5 recombinant proteins were over expressed in Escherichia
coli and purified on a nickel-nitrilotriacetic acid column. Each
of these enzymes was able to reduce phenanthrenequinone, indicating
that they were indeed functional. The activities of these recombinant
proteins were determined as described under "Experimental
Procedures." PGFS1, PGFS2, and DDBX were able to reduce
PGH2 (30 µM) at a rate of 2.5-10 nmol/min
(per mg of enzyme). PGFSL1 and PGFSL2 did not display any PGFS
activity, whereas AKR1B5 processed PGH2 rapidly at a rate
of 22.7 nmol/min. This last enzyme was also able to reduce
17-hydroxyprogesterone (20 µM) and 17-hydroxypregnenolone (20 µM) at 33.6 and 11.9 nmol/min, respectively. The
Lineweaver-Burke graph (Fig. 7) obtained
for AKR1B5 exhibited a Km for PGH2 of
7.1 µM, a Vmax of about 24 nmol/min (per mg of protein) giving a kcat of
0.86 min Despite the preeminent role of PGF2 As an alternate pathway to generate PGF2 Some genes of the AKR1C family were expressed but only at the beginning
of the cycle. No signal was visible at other periods of the cycle or in
cultured cells. By RT-PCR we observed slightly different results.
PGFS1, PGFS2, and PGFSL1 were no
longer detectable at the beginning of the cycle, whereas
DDBX expression was visible in cultured epithelial cells,
and PGFSL2 was expressed all along the estrous cycle and in
cultured cells. The detection of a positive signal for
PGFSL2 can be explained by the greater sensitivity of RT-PCR
analysis. We observed no signal for PGFS1, PGFS2,
and PGFSL1 by PCR at the beginning of the cycle. These
results, contrasting with Northern blot analysis, can be related to
probe cross-hybridization. Indeed, the PGFS1 and
PGFSL1 cDNA probes may have hybridized with the
DDBX and/or PGFSL2 mRNA. This hypothesis is
highly probable because they share long stretches of common nucleic
acid sequences; PGFS1 and PGFSL2 have only one
mismatch between position 490 and 587. The same is true for
PGFSL1 and PGFSL2 between positions 824 and 916 and so on. It is likely that the PGFS cDNA probe bound to the
PGFSL2 mRNA present on the membrane, even under the high stringency conditions used. Moreover, higher expression of
DDBX (and PGFSL2) at the beginning of the cycle
may be related to the presence of leukocytes invading the endometrium
at that moment. The endometrium, collected by scraping, is likely to
contain some leukocytes that may express this gene at a high level.
The present results appear to be in contradiction with those previously
published. Xiao et al. (25, 26) observed variation of the
PGFS1 messenger in cultured cells in response to oxytocin, interferon
From these results, it is doubtful that the strong prostaglandin F
synthase activity present in the bovine endometrium would come from the
PGF synthases PGFS1, PGFS2, or DDBX. Indeed, if we take into account
that stimulated epithelial cells can produce at least 1000 pg of
PGF2 The absence of expression of the known PGFS lead us to search for an
alternate enzyme as described under "Results." We were fortunate
that the AKR1B5 candidate qualified as the PGFS of the bovine
endometrium. First, its mRNA was expressed in endometrial tissues
and cells in relation with the ability of these sources to produce
PGF2 Gene expression of AKR1B5 peaks around day 12, and at the same moment,
progesterone reaches its highest systemic concentration, suggesting a
link between the two. AKR1B5 may be directly or indirectly up-regulated
by progesterone. Because AKR1B5 will also inactivate progesterone, at
least locally, by transforming it into 20 We propose the following model incorporating AKR1B5 in the estrous
cycle. First, ovulation marks the beginning of estrous cycle and the
corpus luteum grows to produce progesterone. Progesterone secretion
peaks between days 12 and 18, and concomitantly AKR1B5 expression rises
in the endometrium. At some point, there is enough AKR1B5 protein to
locally shift the influx:degradation balance toward degradation of
progesterone, hence decreasing the local progesterone concentration.
Then the oxytocin receptor concentration increases. Around day 18, the
first wave of oxytocin occurs, and interaction with its receptor is
possible because the local concentration of progesterone has been
reduced. This interaction leads to activation of phospholipases (40,
41) and subsequently to an increase in PGH2 concentration.
PGH2 is then transformed by AKR1B5 into PGF2 The ability to combine two converging functions, inactivation of
progesterone and generation of PGF2 In summary, we have found that AKR1C family members (to which all the
currently known PGFS belong) are not expressed in the bovine
endometrium. Instead, an aldose reductase known for its 20
(PGF2
) is involved in labor and is the functional
mediator of luteolysis to initiate a new estrous cycle in many species.
These actions have been extensively studied in ruminants, but the
enzymes involved are not clearly identified. Our objective was to
identify which prostaglandin F synthase is involved and to study its
regulation in the endometrium and in endometrial primary cell cultures.
The expression of all previously known prostaglandin F synthases
(PGFSs), two newly discovered PGFS-like genes, and a
20
-hydroxysteroid dehydrogenase was studied by Northern blot and
reverse transcription PCR. These analyses revealed that none of the
known PGFS or the PGFS-like genes were significantly expressed in the
endometrium. On the other hand, the 20
-hydroxysteroid dehydrogenase
gene was strongly expressed in the endometrium at the time of
luteolysis. The corresponding recombinant enzyme has a
Km of 7 µM for PGH2 and a
PGFS activity higher than the lung PGFS. This enzyme has two different activities with the ability to terminate the estrous cycle; it metabolizes progesterone and synthesizes PGF2
. Taken
together, these data point to this newly identified enzyme as the
functional endometrial PGFS.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(PGF2
) is involved in several
physiological processes including pressure regulation in the eye (1), vasoconstriction (2), and renal filtration (3). It is associated with
diseases such as diabetes (4), osteoporosis (5, 6), and menstrual
disorder (7). However, it is mostly known for its effect on the female
reproductive system. In mice, gene knockout of the FP receptor
(the receptor for PGF2
) leads to a failure in the
initiation of labor (8). For most mammalian species, the production of
PGF2
by the uterus is involved in the regulation of the
ovarian cycle. This prostaglandin acts on the corpus luteum, initiating
its regression (luteolysis) and leading to termination of the estrous
cycle or of pregnancy (reviewed in Ref. 9). The regulation of
PGF2
production at the critical period of luteolysis or
recognition of pregnancy has been studied extensively in ruminants. In
cattle, PGF2
is mainly synthesized by epithelial cells
of the endometrium (10). On days 17-20 of the bovine estrous cycle,
oxytocin initiates the release of large pulsatile waves of
PGF2
responsible for the regression of the corpus luteum and the subsequent decrease in progesterone. Despite their primary involvement in the regulation of fertility, the mechanisms involved in
the production of PGF2
at the cellular and molecular
levels are poorly documented.
can be produced from three distinct pathways (see
Fig. 1). The most likely pathway to ensure selective production of
PGF2
results from the reduction of PGH2 by a
9,11-endoperoxide reductase (now referred to as PGFS activity).
Alternate pathways involve the reduction of PGD2 by a
PGD2 11-ketoreductase or the reduction of PGE2
by a 9-ketoprostaglandin reductase (9K-PGR). Previous results obtained
in vitro lead us to believe that the latter pathway could
contribute to the production of PGF2
in cattle. In
support of that hypothesis, we have identified a potential 9K-PGR in
bovine endometrium (11).
,11
PGF2,
an isomer of PGF2
(20). Bovine PGFS1 has
Km values of 120 µM for
PGD2 and 10 µM for PGH2 (14).
DDBX possesses Km values of 10 µM for
PGD2 and 25 µM for PGH2 (14). The
different bovine PGFS are closely related. The PGFS1 and PGFS2 enzymes
are 99% identical, whereas DDBX is 86% identical to both PGFS1 and PGFS2.
-hydroxysteroid dehydrogenase activity accounts for 30%
of soluble protein in the ovaries (21, 22). It is also present at a
lesser extent in the corpus luteum. 9K-PGR activity has also been
observed in the ovine endometrium and corpus luteum (23) and in the
bovine placenta (24). Recently, we have identified a potential 9K-PGR
in bovine endometrium (11). The partial sequence of this putative
enzyme showed 92% homology with bovine lung type prostaglandin F
synthase. For simplicity, we will refer to this enzyme as PGFSL1 for
"prostaglandin F synthase-like 1."
had little influence on the
level of PGFS1 mRNA expression (less than 50% variation), despite
a large effect on prostaglandin production. At parturition in the
sheep, the level of PGFS1 mRNA did not vary in endometrium, myometrium, or placenta (17). Moreover, in all of these experiments, variations in the level of Cox-2 (prostaglandin H synthase 2) mRNA
expression were observed. Thus, it was suggested that either Cox-2
alone was responsible for the increased production of
PGF2
or that the PGFS responsible for the production of
PGF2
in the endometrium was a different, yet
unidentified enzyme.
in the endometrium. The measurement of mRNA
levels in the bovine endometrium of the three known PGF2
synthesizing enzymes (PGFS1, PGFS2, and DDBX) and three newly
identified ones (PGFSL1, PGFSL2, and 20
-HSD) were done by Northern
blotting and RT-PCR throughout the estrous cycle and in cultured
endometrial cells. Recombinant proteins were produced and used to
measure PGFS activity for each candidate enzyme.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
was kindly
provided by Dr. Fuller W. Bazer. [3H]arachidonic acid,
[3H]PGE2, [3H]PGD2,
[3H]PGF2
, the Ready-to-Go T-primed first
strand kit, PCR enzymes (recombinant taq) and buffer, the T7 DNA
polymerase sequencing kit,and the Ready-to-Go cDNA labeling kit
were purchased from Amersham Biosciences. The Ultrasphere C18 column
was purchased from Beckman (Fullerton, CA). The TOPO TA-Cloning pCR2.1
cloning kit was acquired from Invitrogen. The oligonucleotides were
synthesized with a DNA Synthesizer ABI-394 from Applied Biosystem Inc.
Renaissance Western blot chemiluminescence reagent,
[
-32P]UTP, [
-35S]dATP,
[
-32P]dCTP, and [
-32P]ATP were bought
from PerkinElmer Life Sciences. T7 RNA polymerase and RQ1 DNase were
obtained from Promega (Madison, WI). Proteinase K was purchased from
Roche Molecular Biochemicals. RNase T1 and A were obtained from
Pharmingen (San Diego, CA). Nylon membranes were obtained from
Schleicher & Schuell. PGH2 was obtained from Larodan Fine
Chemicals AB (Stockholm, Sweden). Nitrocellulose membrane was purchased
from Bio-Rad. Nickel-nitrilotriacetic acid resin and pET16b plasmids
were bought from Novagene (Madison, WI). Goat anti-rabbit antibody was
obtained from Dako Diagnostics Canada Inc. (Mississauga, Canada). The
Vectastain elite ABC kit was obtained from Vector Laboratories Inc.
(Burlingame, CA). The HPLC apparatus was a Shimadzu system from
Man-tech Associates Inc. (Guelph, Canada). Sequence comparison was made
with the Clustal X software v1.8 (27). Quantification was made using
the
-imager apparatus and software from Alpha Innotech Corp. (San
Leandro, CA).
stimulations were done at 100 nM and 20 µg/ml, respectively.
Oligonucleotides used in the characterization of potential
prostaglandin F synthase in the bovine endometrium
-32P]dCTP (3000 Ci/mmol) using a
cDNA labeling kit. 15 µg of total RNA were loaded on each lane of
a 1% agarose/formaldehyde gel and blotted after electrophoresis on a
nylon membrane (Nytran Plus). Hybridization with the appropriate
radiolabeled probe was performed overnight at 42 °C in a 50%
formamide solution. The washings were done in 0.2% SSC at 65 °C,
and the membranes were exposed to a Biomax film (Amersham Biosciences)
at
80 °C until good signals were observed.
-actin was
performed with oligonucleotides BACTINs and BACTINas for 30 cycles with an annealing temperature of 55 °C yielding a fragment of 349 bp. All
of the amplified PCR products were sequenced and estimated to span over
several introns by reference with corresponding human genes found
in GenBankTM.
-actin were made by inserting amplified PCR fragments into TOPO
pCR2.1 as described above. Plasmids containing AKR1B5 were digested
with XcmI and transcribed with T7 RNA polymerase to yield a
full-length RNA probe of 441 nucleotides comprising 339 nucleotides
complementary (protected) to ARK1B5 mRNA. Plasmids containing
-actin were digested with BglI and transcribed with T7
RNA polymerase to yield a full-length RNA probe of 242 nucleotides
comprising 170 nucleotides complementary to
-actin mRNA. The
riboprobe synthesis and RNase protection assay were performed according
to Pharmingen standard RPA procedure.
was confirmed by TLC
using silica plates and enzyme-linked immunosorbent assay. Migration was performed in ethyl acetate:2,2,4-trimethylpentane:acetic acid (110:50:20) water-saturated solvent, and detection was achieved by
spraying phosphomolybdic acid 10% (w/v) in methanol and cooking the
plate at 120 °C for 10 min (30).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Biosynthesis--
We first determined whether
PGF2
could be synthesized from PGH2,
PGE2, or PGD2 (Fig.
1) in endometrial cells. Epithelial cells
were treated with oxytocin, a treatment known to specifically increase
the production of PGF2
(32) and supplied with [3H]arachidonic acid, [3H]PGE2,
or [3H]PGD2. 24 h later, the radioactive
prostaglandins present in the supernatant were analyzed by HPLC (Fig.
2). As expected,
[3H]PGF2
is the main prostaglandin
produced when tritiated arachidonic acid is given to the cells. No
radioactive PGF2
was produced when
[3H]PGE2 or
[3H]PGD2 were given to the cells, indicating
that no 9-ketoreductase or 11-ketoreductase activity was present in
cultured endometrial epithelial cells. The absence of 11-ketoreductase
activity was surprising because all known PGFS exhibit this activity.
The product of PGD2 reduction, 9
,11
PGF2,
was shown to elute almost at the same position as PGF2
(33). The unknown radioactive products appearing in 2B and 2C might be
related to prostaglandin catabolism.
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Fig. 1.
Known biosynthetic pathways leading to the
formation of PGF2 .
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Fig. 2.
Prostaglandin production from different
radiolabeled precursors in oxytocin-treated endometrial epithelial
cells in primary culture. The analysis was performed by HPLC on a
C18 column using an acetonitrile:water:acetic acid (45:55:0.05)
isocratic elution. For each experiment, the peak assignment was done
with tritiated prostaglandin standards (not shown). Slight variations
in retention time are due to pump leakage.
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Fig. 3.
Northern blot analysis of PGFS (1 and 2),
DDBX, PGFSL1, and PGFSL2 throughout the estrous cycle in the
endometrium. Lanes 1, days 1-4; lanes 2,
days 5-8; lanes 3, days 9-12; lanes 4, days
13-15; lanes 5, days 16-18; lanes 6, days
19-21. Lu, lung; Li, liver; Ep,
epithelial cells (treated with oxytocin for 6 h); St,
stromal cells. Exposure was for one week. 18 S ribosomal RNA is shown
as loading controls.
-actin-positive control was amplified in all
cases. RT-PCR analysis was also conducted for PGFSL1 and
PGFSL2 (Fig. 4, panels iii and iv).
For PGFSL1, the signal was present only in liver. For
PGFSL2, a signal was present throughout with stronger
expression in the liver and in the endometrium at the beginning of the
cycle. The identities of all amplified products were confirmed by
sequencing.
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Fig. 4.
Expression of the three known and the two
potential bovine PGF synthase genes. PGFS1, PGFS2, and DDBX
expression was studied by RT-PCR on total RNA in the endometrium
(i) and in epithelial cell cultures (ii).
Amplification of -actin was used as a control. RT-PCR analysis was
also performed for PGFSL1 (iii) and PGFSL2 (iv)
throughout the estrous cycle in the endometrium and in cell cultures.
Lanes 1, days 1-4; lanes 2, days 5-8;
lanes 3, days 9-12; lanes 4, days 13-15;
lanes 5, days 16-18; lanes 6, days 19-21.
Li, liver; Lu, lung; E, untreated
epithelial cells; EO, epithelial cells treated with oxytocin
for 6 h; ET, epithelial cells treated with interferon
for 6 h; S, stromal cells.
is present in the bovine
endometrium in absence of any known PGFS. Because all of these PGFS
belong to the AKR1C family and shared at least 80% homology, we have
designed oligonucleotide probes in regions common to all AKR1C family
members. Northern analysis of endometrial mRNA revealed that no
AKR1C family member was expressed at times of high PGF2
production (34). An alternate pathway to produce PGF2
is
through 9-ketoreductase conversion of PGE2. A former
candidate for PGF2
production in the endometrium, the
rabbit 9-ketoreductase, also exhibit 20
-HSD activity (21, 22).
Therefore, we searched GenBankTM to find whether there was
any aldoketoreductase with 20
-HSD activity that was known in cattle.
Interestingly, we found an aldose reductase identified as AKR1B5,
having only 45% homology with the PGFSs of the AKR1C family but
expressing 20-
hydroxysteroid dehydrogenase activity (35). To
investigate the potential role of this enzyme, we designed specific
oligonucleotides and found that its gene was highly expressed during
the estrous cycle and in endometrial cell cultures (data not shown) at
times just preceding the expected production of PGF2
.
Northern blot, RT-PCR, and RNase protection analysis revealed that
AKR1B5 gene was highly expressed in the endometrium from days 10 to 21 (Fig. 5). Panels A,
B, and D of Fig. 5 represent three distinct sets
of samples, and slight variations in expression may depend on
individual variations. The identity of the amplified products was
confirmed by sequence analysis. RNase protection assay confirmed
unequivocally that AKR1B5 is expressed in the endometrium and modulated
throughout the estrous cycle. We cloned and produced the recombinant
protein to generate antibodies needed to characterize this enzyme.
Western blot analysis showed that the protein followed a
similar but slightly delayed pattern of expression (Fig.
6, B and C). Fig.
6A indicates that the AKR1B5 antibody is specific to AKR1B5
or at least to the AKR1B family because it can also recognize its human
counterpart AKR1B1 (data not shown). A faint signal was observed for
some AKR1C recombinant proteins, but we had to put 50 times more AKR1C protein to obtain a signal equivalent to that of AKR1B5. We evaluated the amount of AKR1B5 protein present in our sample by comparing its
signal against a standard curve of AKR1B5 recombinant protein (Fig.
6D). Samples from days 4-8 contain about 20 ng (0.2% of the proteins present in the sample) of AKR1B5, and those from days
16-18 contain 200 ng (2%). Thus, if any member of the AKR1C family
was responsible for this signal, it would have to constitute 100% of
the protein in the sample.
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Fig. 5.
Expression of AKR1B5 gene throughout the
estrous cycle. Total RNA was extracted from endometrial scrapings
collected at different days of the estrous cycle as described under
"Experimental Procedures." A, Northern blot analysis
with 15 µg of total RNA/lane. B, RT-PCR.
C, relative expression of AKR1B5 gene (means ± S.D.)
present in four sets of samples evaluated by PCR. D, RNase
protection assay. Signals at 411 and 242 represent the full-length
probe for AKR1B5 and -actin, respectively. Those at 339 and 170 correspond to the portion of the probes protected by AKR1B5 and
-actin mRNA, respectively. Each assay was performed with 1 µg
of total RNA. NS, nonspecific control where yeast RNA was
used. The sequence reaction to the left was used as a
ladder. E, relative expression of AKR1B5 over
-actin is
presented as a ratio of density of bands observed at 339 and 170. Throughout the figure, D followed by number represents the
day of the estrous cycle. 18 S RNA and
-actin were used as loading
controls.
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Fig. 6.
Expression of AKR1B5 protein throughout the
estrous cycle as evaluated by Western blot analysis. A,
the specificity of the AKR1B5 antibody was evaluated with different
recombinant protein of the AKR1C family (60 ng of
protein/lane). B and C represent two
different series of proteins extracted from endometrial scrapings taken
at different days of the estrous cycle as described under
"Experimental Procedures" (10 µg/lane). D,
titration of AKR1B5. 10 µg of endometrial protein from days 4-8 or
16-18 were compared with 10 and 100 ng of purified recombinant AKR1B5.
LC, Coomassie Blue staining used as loading controls.
1 (assuming a 100% active enzyme). The
conversion of PGH2 into PGF2
was confirmed
by TLC (Fig. 7) and by enzyme-linked immunosorbent assay. In absence of
enzyme (C), a spot was found only for PGE2 generated from
spontaneous conversion of PGH2 (36), whereas in the
presence of AKR1B5 (E), a new spot appeared representing PGF2
. None of the enzymes tested displayed any
9-ketoreductase activity. Localization of AKR1B5 enzyme by
immunohistochemistry shows expression in luminal and glandular
epithelial cells (Fig. 8).
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Fig. 7.
PGFS activity of AKR1B5;
Km determination for PGH2.
Top panel, Lineweaver-Burke analysis. Bottom
panel, TLC showing PGF2 formation. C,
control reaction without enzyme. E, reaction in presence of
AKR1B5. M, PGF2
and PGE2
markers.
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Fig. 8.
Localization of AKR1B5 in the endometrium of
a day 17 cow, determined by immunohistochemistry. A,
preimmune serum, dilution 1:750. B, AKR1B5 anti-serum
dilution 1:750. LE, luminal epithelial cells; GE,
glandular epithelial cells; S, stromal cells.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
in reproductive
function in mammals, there has been no formal identification of the biosynthetic enzyme(s) responsible for its selective production. The
bovine endometrium is the main source of PGF2
at the time of luteolysis. The results obtained in vitro in our
laboratory suggested that PGF2
could be produced at
least in part through conversion of PGD2 and/or
PGE2. This possibility is supported by the ability of
cultured epithelial cells to generate PGF2
, PGE2, and PGD2. HPLC analysis of prostaglandin
metabolism (Fig. 2), however, showed no detectable 9-ketoreductase or
11-ketoreductase activity in control or oxytocin-treated epithelial
cells. These two activities were not observed in stromal cells either
(data not shown). Therefore, in these cells, PGF2
production appears to derive mainly by direct PGH2
reduction (Fig. 1). This result was surprising because all PGFS are
known to possess 11-ketoreductase activity. This was the first
indication that in the bovine endometrium, PGF2
is
produced by a PGFS activity distinct from what was observed in other
tissues. Some may argue that the phenomenon observed in cell culture
may vary from what occurs in vivo. This is unlikely, because
the large increase in PGF2
production occurring in
vivo in response to oxytocin at the time of luteolysis can be
reproduced in vitro (32).
, we were able
to complete the previously published partial putative 9K-PGR sequence (PGFSL1) (11). In the process, we were fortunate to find
PGFSL2, another gene highly related to PGFS1.
With DDBX and PGFS2, the AKR1C family now counts
five members in the bovine species. This situation is similar to the
humans where PGFS (AKR1C3) belongs to a group of four highly homologous
enzymes of the same family (AKR1C1, AKR1C2, AKR1C3, and AKR1C4) (19).
Because all the members of this family are highly homologous, the use
of Northern blots and RT-PCR may be misleading, and results must be
interpreted with caution.
, and steroid treatments. These experiments were conducted by
Northern blot analysis with 25 µg of total RNA (compared with 15 µg
in the present study) under low stringency conditions (2× SSC at
55 °C). Under these conditions, the PGFS1 cDNA probe may have
cross-hybridized with any of the five members of the AKR1C family. We
have also detected PGFS1 by RT-PCR in cultured epithelial cells (11). But the primers used then would have given a PCR product of
the same size for PGFS2, PGFSL1, and
PGFSL2. It is likely that in all of these experiments
PGFSL2 was detected instead of PGFS1.
/µg of protein in 24 h and that the known
PGFSs have a specific activity (with 40 µM
PGH2) ranging from 3 to 56 nmol/mg of protein/min (21),
they should represent at least 0.005-0.1% of total proteins. Because
it is unlikely that PGH2 levels reach 40 µM
in the cytosol, the actual enzyme levels would have to be even higher.
Moreover, the absence of mRNA for the known PGFS and the lack of
11-ketoreductase activity in endometrial cells despite a large
production of PGF2
eliminates these enzymes as
functional PGFS in the bovine endometrium. Similarly, the low
expression of PGFSL1 and PGFSL2 combined with the lack of PGFS activity
of their recombinant proteins indicate that neither of these enzymes is
involved in PGF2
formation in the endometrium. These
enzymes may, however, be involved in steroid metabolism because it is
the case for most enzymes of the AKR1C family.
. Second, the recombinant AKR1B5 protein did not
display any 9-ketoprostaglandin reductase activity but a strong PGFS
activity. The Km of AKR1B5 for PGH2 is
half that of the recombinant lung type PGFS (14), and its specific
activity is twice as high. The 20
-HSD activity of our recombinant
protein was present as expected and was comparable with the purified
native enzyme (35).
-hydroxyprogesterone, it
may down-regulate its own expression. Moreover, local metabolism of
progesterone may have a physiological role in the endometrium. In
ovarectomized sheep, progesterone withdrawal causes a build-up in
oxytocin receptors (37). In intact animals, it is thought that
progesterone down-regulates its own receptor, therefore creating a
situation similar to progesterone withdrawal (9). However, Robinson
et al. (38) recently observed that the up-regulation of
oxytocin receptors, occurring around days 15-16, in luminal epithelial
cells was not dependent on a prior change in progesterone or estradiol
receptors. Additionally, Bogacki et al. (39) demonstrated that oxytocin was unable to bind to its own receptor in
the presence of progesterone. This can constitute a major problem to
initiate PGF2
production and luteolysis, because when the initial oxytocin bursts occurs, the organism is flooded with progesterone. It is possible that AKR1B5 reduces local progesterone concentration, allowing oxytocin to act on the endometrium.
. After several waves of PGF2
, the
corpus luteum is destroyed, abolishing the production of progesterone.
The reproductive system is then ready for a new cycle.
, makes AKR1B5 a multifunctional enzyme with complementary action in the endometrium. Moreover, as an aldose reductase, this enzyme can also reduce benzaldehyde, glyceraldehyde, glucose, and several other
carbonyl-containing compounds. This enzyme can be found in a wide
variety of tissues. In humans a corresponding enzyme, AKR1B1, is
involved in some complications associated with diabetes such as eye
disease (cataracts) and nephropathy. It is believed that these
complications are caused by sorbitol accumulation following reduction
of glucose. Because PGF2
is also involved in these
diseases, the possibility that AKR1B1 may act as a PGF synthase in
those organs is worth investigating.
-HSD
activity, AKR1B5, is a likely candidate enzyme for controlling the
sufficient and timely production of PGF2
in the bovine endometrium. This is the first time that a member of the AKR1B family
has been associated with PGFS activity and also the first report of
such an enzyme being expressed in the endometrium of any species.
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FOOTNOTES |
---|
* This work was supported by grants from the Canadian Institutes for Health Research and Natural Sciences and Engineering Research Council of Canada.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of a Wyeth-Ayerst/Canadian Institutes for Health
Research postdoctoral fellowship.
§ To whom correspondence should be addressed: Unité de Recherche en Ontogénie et Reproduction, Centre Hospitalier Universitaire de Québec, Université Laval, 2705, Boul. Laurier, Sainte-Foy, Québec G1V 4G2, Canada. Tel.: 418-656-4141 (ext. 6141); Fax: 418-654-2714; E-mail: mafortier@crchul.ulaval.ca.
Published, JBC Papers in Press, January 24, 2003, DOI 10.1074/jbc.M208318200
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ABBREVIATIONS |
---|
The abbreviations used are:
PG, prostaglandin;
PGHS, PGH synthase;
PGFS, PGF synthase;
9K-PGR, 9-ketoprostaglandin
reductase;
DDBX, dihydrodiol dehydrogenase 3;
20-HSD, 20
-hydroxysteroid dehydrogenase;
RT, reverse transcriptase;
HPLC, high pressure liquid chromatography;
RACE, rapid
amplification of cDNA ends.
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REFERENCES |
---|
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---|
1. | Crawford, K., and Kaufman, P. L. (1987) Arch. Ophthalmol. 105, 1112-1116[Abstract] |
2. | Cracowski, J. L., Devillier, P., Durand, T., Stanke-Labesque, F., and Bessard, G. (2001) J. Vasc. Res. 38, 93-103[CrossRef][Medline] [Order article via Infotrieve] |
3. | Weber, P. C. (1980) Contrib. Nephrol. 23, 83-92[Medline] [Order article via Infotrieve] |
4. | Mezzetti, A., Cipollone, F., and Cuccurullo, F. (2000) Cardiovasc. Res. 47, 475-488[CrossRef][Medline] [Order article via Infotrieve] |
5. | Ma, Y. F., Li, X. J., Jee, W. S. S., Mcosker, J., Liang, X. G., Setterberg, R., and Chow, S. Y. (1995) Bone 17, 549-554[CrossRef][Medline] [Order article via Infotrieve] |
6. | Soper, D. L., Milbank, J. B., Mieling, G. E., Dirr, M. J., Kende, A. S., Cooper, R., Jee, W. S., Yao, W., Chen, J. L., Bodman, M., Lundy, M. W., De, B., Stella, M. E., Ebetino, F. H., Wang, Y., deLong, M. A., and Wos, J. A. (2001) J. Med. Chem. 44, 4157-4169[CrossRef][Medline] [Order article via Infotrieve] |
7. | Poyser, N. L. (1995) Prostaglandins Leukotrienes Essent. Fatty Acids 53, 147-195[Medline] [Order article via Infotrieve] |
8. |
Sugimoto, Y.,
Yamasaki, A.,
Segi, E.,
Tsuboi, K.,
Aze, Y.,
Nishimura, T.,
Oida, H.,
Yoshida, N.,
Tanaka, T.,
Katsuyama, M.,
Hasumoto, K.,
Murata, T.,
Hirata, M.,
Ushikubi, F.,
Negishi, M.,
Ichikawa, A.,
and Narumiya, S.
(1997)
Science
277,
681-683 |
9. |
McCracken, J. A.,
Custer, E. E.,
and Lamsa, J. C.
(1999)
Physiol. Rev.
79,
263-323 |
10. | Fortier, M. A., Guilbault, L. A., and Grasso, F. (1988) J. Reprod. Fertil. 83, 239-248[Abstract] |
11. |
Asselin, E.,
and Fortier, M. A.
(2000)
Biol. Reprod.
62,
125-131 |
12. |
Watanabe, K.,
Yoshida, R.,
Shimizu, T.,
and Hayaishi, O.
(1985)
J. Biol. Chem.
260,
7035-7041 |
13. | Kuchinke, W., Barski, O., Watanabe, K., and Hayaishi, O. (1992) Biochem. Biophys. Res. Commun. 183, 1238-1246[Medline] [Order article via Infotrieve] |
14. |
Suzuki, T.,
Fujii, Y.,
Miyano, M.,
Chen, L. Y.,
Takahashi, T.,
and Watanabe, K.
(1999)
J. Biol. Chem.
274,
241-248 |
15. | Chen, L. Y., Watanabe, K., and Hayaishi, O. (1992) Arch. Biochem. Biophys. 296, 17-26[Medline] [Order article via Infotrieve] |
16. | Suzuki-Yamamoto, T., Nishizawa, M., Fukui, M., Okuda-Ashitaka, E., Nakajima, T., Ito, S., and Watanabe, K. (1999) FEBS Lett. 462, 335-340[CrossRef][Medline] [Order article via Infotrieve] |
17. | Wu, W. X., Ma, X. H., Yoshizato, T., Shinozuka, N., and Nathanielsz, P. W. (2001) J. Soc. Gynecol. Invest. 8, 69-76[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Kubata, B.,
Duszenko, M.,
Kabututu, Z.,
Rawer, M.,
Szallies, A.,
Fujimori, K.,
Inui, T.,
Nozaki, T.,
Yamashita, K.,
Horii, T.,
Urade, Y.,
and Hayaishi, O.
(2000)
J. Exp. Med.
192,
1327-1338 |
19. | Jez, J. M., Flynn, T. G., and Penning, T. M. (1997) Biochem. Pharmacol. 54, 639-664[CrossRef][Medline] [Order article via Infotrieve] |
20. | Watanabe, K., Iguchi, Y., Iguchi, S., Arai, Y., Hayaishi, O., and Roberts, L. J., II (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 1583-1587[Abstract] |
21. | Lacy, W. R., Washenick, K. J., Cook, R. G., and Dunbar, B. S. (1993) Mol. Endocrinol. 7, 58-66[Abstract] |
22. | Wintergalen, N., Thole, H. H., Galla, H. J., and Schlegel, W. (1995) Eur. J. Biochem. 234, 264-270[Abstract] |
23. | Beaver, C. J., and Murdoch, W. J. (1992) Prostaglandins 44, 37-42[CrossRef][Medline] [Order article via Infotrieve] |
24. | Kankofer, M., and Wiercinski, J. (1999) Prostaglandins Leukotrienes Essent. Fatty Acids 61, 29-32[CrossRef][Medline] [Order article via Infotrieve] |
25. |
Xiao, C. W.,
Liu, J. M.,
Sirois, J.,
and Goff, A. K.
(1998)
Endocrinology
139,
2293-2299 |
26. |
Xiao, C. W.,
Murphy, B. D.,
Sirois, J.,
and Goff, A. K.
(1999)
Biol. Reprod.
60,
656-663 |
27. | Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F., and Higgins, D. G. (1997) Nucleic Acids Res. 24, 4876-4882[CrossRef] |
28. |
Arosh, J. A.,
Parent, J.,
Chapdelaine, P.,
Sirois, J.,
and Fortier, M. A.
(2002)
Biol. Reprod.
67,
161-169 |
29. | Asselin, E., Goff, A. K., Bergeron, H., and Fortier, M. A. (1996) Biol. Reprod. 54, 371-379[Abstract] |
30. | Gréen, K., Hamberg, M., Samuelsson, B., and Frölich, J. C. (1978) in Advances in Prostaglandin and Thromboxane Research (Frölich, J. C., ed), Vol. 5 , pp. 15-24, Raven Press, New York |
31. | Chapdelaine, P., Vignola, K., and Fortier, M. A. (2001) BioTechniques 31, 478[Medline] [Order article via Infotrieve], 480, and 482 |
32. |
Asselin, E.,
Drolet, P.,
and Fortier, M. A.
(1997)
Endocrinology
138,
4798-4805 |
33. |
Urade, Y.,
Watanabe, K.,
Eguchi, N.,
Fujii, Y.,
and Hayaishi, O.
(1990)
J. Biol. Chem.
265,
12029-12035 |
34. | Madore, E., Arosh, J. A., Parent, J., Villeneuve, C., Chapdelaine, P., and Fortier, M. A. (2001) Biol. Reprod. 64 (Suppl. 1), 319 |
35. | Warren, J. C., Murdock, G. L., Ma, Y., Goodman, S. R., and Zimmer, W. E. (1993) Biochemistry 32, 1401-1406[Medline] [Order article via Infotrieve] |
36. |
Jakobsson, P. J.,
Thoren, S.,
Morgenstern, R.,
and Samuelsson, B.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
7220-7225 |
37. | Leavitt, W. W., Okulicz, W. C., McCracken, J. A., Schramm, W., and Robidoux, W. F., Jr (1985) J. Steroid Biochem. 22, 687-691[CrossRef][Medline] [Order article via Infotrieve] |
38. |
Robinson, R. S.,
Mann, G. E.,
Lamming, G. E.,
and Wathes, D. C.
(2001)
Reproduction
122,
965-979 |
39. |
Bogacki, M.,
Silvia, W. J.,
Rekawiecki, R.,
and Kotwica, J.
(2002)
Biol. Reprod.
67,
184-188 |
40. | Lee, J. S., and Silvia, W. J. (1994) J. Endocrinol. 141, 491-496[Abstract] |
41. | Silvia, W. J., Lee, J. S., Trammell, D. S., Hayes, S. H., Lowberger, L. L., and Brockman, J. A. (1994) J. Endocrinol. 141, 481-490[Abstract] |