An Aldose Reductase with 20alpha -Hydroxysteroid Dehydrogenase Activity Is Most Likely the Enzyme Responsible for the Production of Prostaglandin F2alpha in the Bovine Endometrium*

Eric MadoreDagger, Nathalie Harvey, Julie Parent, Pierre Chapdelaine, Joe A. Arosh, and Michel A. Fortier§

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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Prostaglandins are important regulators of reproductive function. In particular, prostaglandin F2alpha (PGF2alpha ) 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 20alpha -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 20alpha -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 PGF2alpha . Taken together, these data point to this newly identified enzyme as the functional endometrial PGFS.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 F2alpha (PGF2alpha ) 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 PGF2alpha ) leads to a failure in the initiation of labor (8). For most mammalian species, the production of PGF2alpha 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 PGF2alpha production at the critical period of luteolysis or recognition of pregnancy has been studied extensively in ruminants. In cattle, PGF2alpha 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 PGF2alpha 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 PGF2alpha at the cellular and molecular levels are poorly documented.

PGF2alpha can be produced from three distinct pathways (see Fig. 1). The most likely pathway to ensure selective production of PGF2alpha 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 PGF2alpha in cattle. In support of that hypothesis, we have identified a potential 9K-PGR in bovine endometrium (11).

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 9alpha ,11beta PGF2, an isomer of PGF2alpha (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.

9-Ketoprostaglandin reductase activity has been detected in the reproductive system of several species. In the rabbit, a 9K-PGR that possesses 20alpha -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."

Studies on the regulation of PGFS1 have already been performed on cultured bovine endometrial cells (11, 25, 26). Treatments with hormones, oxytocin, and interferon tau  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 PGF2alpha or that the PGFS responsible for the production of PGF2alpha in the endometrium was a different, yet unidentified enzyme.

The objective of the present work was to identify the metabolic pathways and the enzymes involved in the formation of PGF2alpha in the endometrium. The measurement of mRNA levels in the bovine endometrium of the three known PGF2alpha synthesizing enzymes (PGFS1, PGFS2, and DDBX) and three newly identified ones (PGFSL1, PGFSL2, and 20alpha -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.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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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 tau  was kindly provided by Dr. Fuller W. Bazer. [3H]arachidonic acid, [3H]PGE2, [3H]PGD2, [3H]PGF2alpha , 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, [alpha -32P]UTP, [alpha -35S]dATP, [alpha -32P]dCTP, and [gamma -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 alpha -imager apparatus and software from Alpha Innotech Corp. (San Leandro, CA).

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 tau  stimulations were done at 100 nM and 20 µg/ml, respectively.

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).


                              
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Table I
Oligonucleotides used in the characterization of potential prostaglandin F synthase in the bovine endometrium

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 [alpha -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.

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 beta -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.

RNase Protection Assay-- Antisense riboprobes for AKR1B5 and beta -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 beta -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 beta -actin mRNA. The riboprobe synthesis and RNase protection assay were performed according to Pharmingen standard RPA procedure.

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 PGF2alpha 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).

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.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

PGF2alpha Biosynthesis-- We first determined whether PGF2alpha 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 PGF2alpha (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]PGF2alpha is the main prostaglandin produced when tritiated arachidonic acid is given to the cells. No radioactive PGF2alpha 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, 9alpha ,11beta PGF2, was shown to elute almost at the same position as PGF2alpha (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 PGF2alpha .


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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.

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).


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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.

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 beta -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 beta -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 tau  for 6 h; S, stromal cells.

Identification of an Alternate PGFS-- These results show that of a massive production of PGF2alpha 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 PGF2alpha production (34). An alternate pathway to produce PGF2alpha is through 9-ketoreductase conversion of PGE2. A former candidate for PGF2alpha production in the endometrium, the rabbit 9-ketoreductase, also exhibit 20alpha -HSD activity (21, 22). Therefore, we searched GenBankTM to find whether there was any aldoketoreductase with 20alpha -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-alpha 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 PGF2alpha . 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 beta -actin, respectively. Those at 339 and 170 correspond to the portion of the probes protected by AKR1B5 and beta -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 beta -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 beta -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.

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-1 (assuming a 100% active enzyme). The conversion of PGH2 into PGF2alpha 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 PGF2alpha . 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 PGF2alpha formation. C, control reaction without enzyme. E, reaction in presence of AKR1B5. M, PGF2alpha 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.


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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

Despite the preeminent role of PGF2alpha 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 PGF2alpha at the time of luteolysis. The results obtained in vitro in our laboratory suggested that PGF2alpha 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 PGF2alpha , 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, PGF2alpha 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, PGF2alpha 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 PGF2alpha production occurring in vivo in response to oxytocin at the time of luteolysis can be reproduced in vitro (32).

As an alternate pathway to generate PGF2alpha , 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.

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 tau , 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.

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 PGF2alpha /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 PGF2alpha 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 PGF2alpha 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.

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 PGF2alpha . 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 20alpha -HSD activity of our recombinant protein was present as expected and was comparable with the purified native enzyme (35).

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 20alpha -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 alpha  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 PGF2alpha 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.

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 PGF2alpha . After several waves of PGF2alpha , the corpus luteum is destroyed, abolishing the production of progesterone. The reproductive system is then ready for a new cycle.

The ability to combine two converging functions, inactivation of progesterone and generation of PGF2alpha , 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 PGF2alpha is also involved in these diseases, the possibility that AKR1B1 may act as a PGF synthase in those organs is worth investigating.

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 20alpha -HSD activity, AKR1B5, is a likely candidate enzyme for controlling the sufficient and timely production of PGF2alpha 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.

    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.

Dagger 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

    ABBREVIATIONS

The abbreviations used are: PG, prostaglandin; PGHS, PGH synthase; PGFS, PGF synthase; 9K-PGR, 9-ketoprostaglandin reductase; DDBX, dihydrodiol dehydrogenase 3; 20alpha -HSD, 20alpha -hydroxysteroid dehydrogenase; RT, reverse transcriptase; HPLC, high pressure liquid chromatography; RACE, rapid amplification of cDNA ends.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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