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Prostaglandin Metabolism in the Ovine Corpus Luteum: Catabolism of Prostaglandin F₂ (PGF₂) Coincides with Resistance of the Corpus Luteum to PGF₂¹

^a Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, Colorado 80526

ABSTRACT

To examine possible mechanisms involved in resistance of the ovine corpus luteum to the luteolytic activity of prostaglandin (PG)F₂, the enzymatic activity of 15-hydroxyprostaglandin dehydrogenase (PGDH) and the quantity of mRNA encoding PGDH and cyclooxygenase (COX-2) were determined in ovine corpora lutea on Days 4 and 13 of the estrous cycle and Day 13 of pregnancy. The corpus luteum is resistant to the action of PGF₂ on Days 4 of the estrous cycle and 13 of pregnancy while on Day 13 of the estrous cycle the corpus luteum is sensitive to the actions PGF₂. Enzymatic activity of PGDH, measured by rate of conversion of PGF₂ to PGFM, was greater in corpora lutea on Day 4 of the estrous cycle (P < 0.05) and Day 13 of pregnancy (P < 0.05) than on Day 13 of the estrous cycle. Levels of mRNA encoding PGDH were also greater in corpora lutea on Day 4 of the estrous cycle (P < 0.01) and Day 13 of pregnancy (P < 0.01) than on Day 13 of the estrous cycle. Thus, during the early estrous cycle and early pregnancy, the corpus luteum has a greater capacity to catabolize PGF, which may play a role in the resistance of the corpus luteum to the actions of this hormone. Levels of mRNA encoding COX-2 were undetectable in corpora lutea collected on Day 13 of the estrous cycle but were 11 ± 4 and 44 ± 28 amol/µg poly(A)⁺ RNA in corpora lutea collected on Day 4 of the estrous cycle and Day 13 of pregnancy, respectively. These data suggest that there is a greater capacity to synthesize PGF₂, early in the estrous cycle and early in pregnancy than on Day 13 of the estrous cycle. In conclusion, enzymatic activity of PGDH may play an important role in the mechanism involved in luteal resistance to the luteolytic effects of PGF₂.

corpus luteum, corpus luteum function

INTRODUCTION

The ovine corpus luteum is required for survival and growth of the embryo through the first 50 days of pregnancy [1, 2]. The corpus luteum of pregnancy is rescued from the luteolytic effects of prostaglandin (PG)F₂ by 1) the actions of interferon (IFN)- [3, 4] to suppress pulsatile secretion of PGF₂ from the uterus and 2) establishment of mechanisms that promote resistance of the corpus luteum to the luteolytic effects of PGF₂ [5–9].

The biochemical mechanisms involved in luteal resistance to PGF₂ are not well described, but interference with the second messenger pathway of PGF₂ action is believed to be involved [10]. On Day 4 of the estrous cycle, when the corpus luteum is resistant to PGF₂, levels of mRNA encoding two endogenous peptide inhibitors of protein kinase C are elevated [10]. Downregulation of receptors for PGF₂ does not appear to be a mechanism because expression of receptors [11] or mRNA encoding receptors for PGF₂ [10] are not attenuated during maternal recognition of pregnancy in ewes or cows [12]. In fact, levels of mRNA encoding the receptor for PGF₂ are elevated on Day 14 in pregnant ewes relative to nonpregnant ewes [13]. Furthermore, the affinity of receptors for PGF₂ does not change during the estrous cycle or during maternal recognition of pregnancy in cows [12] and ewes [11]. Thus, the mechanism by which the ruminant corpus luteum achieves resistance to the luteolytic activity of PGF₂ does not appear to involve regulation of PGF₂ receptors.

It is possible that catabolism of PGF₂ by the corpus luteum is involved in the resistance of the corpus luteum of early pregnancy to PGF₂. The enzyme that performs the rate-limiting enzymatic step in inactivation of prostaglandins of the E and F series is 15-hydroxyprostaglandin dehydrogenase (PGDH) [14–16]. Many tissues express PGDH to provide local protection from the biological effects of PGF₂. For example, the human chorion expresses high levels of PGDH to provide a barrier to PGF₂ (of amnionic origin) [17, 18] that could reach the myometrium. The lungs of pregnant rabbits increase expression of PGDH in response to progesterone [19, 20], perhaps to provide protection of the corpora lutea of pregnancy from circulating PGF₂ [21].

Resistance of the corpus luteum could also be facilitated by attenuation of production of PGF₂ by the corpus luteum. On Day 4 of the estrous cycle in the cow, when the corpus luteum is resistant to the luteolytic activity of PGF₂, this hormone does not stimulate increases in the level of mRNA encoding cyclooxygenase-2 (COX-2) [22]. However, on Day 10 of the estrous cycle, when the corpus luteum is sensitive to the luteolytic effects of PGF, levels of mRNA encoding COX-2 are elevated in response to PGF₂ [22]. The rate-limiting step in biosynthesis of prostanoids is catalyzed by COX-2; therefore, an increase in levels of mRNA encoding COX-2 might facilitate an increase in luteal synthesis of PGF₂, establishing an ultrashort positive feedback loop that culminates in luteolysis.

The first objective of this study was to determine if enzymatic activity of PGDH in corpora lutea during early pregnancy and the early estrous cycle, when luteal resistance to the luteolytic effects of PGF₂ is known to occur, was greater than the activity during the late luteal phase of the estrous cycle. If luteal synthesis of PGF₂ plays a role in luteolysis, then reduced activity of the key enzyme in this pathway, COX-2 could also be critical for luteal resistance to PGF₂. Therefore, the second objective was to determine if expression of mRNA encoding COX-2 was suppressed during periods when the corpus luteum is resistant to the luteolytic effects of PGF₂.

MATERIALS AND METHODS

Unless otherwise indicated, all reagents and materials were obtained from Sigma Chemical (St. Louis, MO) or Fisher Scientific (Denver, CO).

Experimental Design

To test the hypothesis that expression and enzymatic activity of PGDH and expression of COX-2 in ovine corpora lutea is greater on Day 4 postestrus in nonpregnant ewes and Day 13 postcoitus in pregnant ewes than on Day 13 postestrus in nonpregnant ewes, 28 ewes were randomly assigned to three groups (Day 4 of the estrous cycle, n = 11; Day 13 of the estrous cycle, n = 7; Day 13 of pregnancy, n = 10). Based on the mean and variability figures for PGDH activity measurements in previous experiments [23], power calculations indicated that a sample size of seven ewes was necessary to detect differences in PGDH activity with a power of 0.96. Because of the minute amount of tissue in the Day 4 corpus luteum, tissue was collected from 11 ewes to increase the probability of collecting multiple corpora lutea from a single ewe to insure that enough tissue would be available for all analyses without pooling tissue and compromising the experimental unit (the ewe). Similarly, 10 ewes were bred for collection of corpora lutea on Day 13 of pregnancy in anticipation of 70% pregnancy rates; 10 ewes became pregnant so luteal tissue was collected from each animal.

Tissue Collection

All experimental procedures and protocols were reviewed and approved by the Colorado State University Animal Care and Use Committee. Western range ewes exhibiting normal estrous cycles (17 ± 1 days) were checked for estrous behavior twice daily by a vasectomized ram, and the first day a ewe was observed in estrus was designated as Day 0 of the estrous cycle. To collect tissue from ewes on Day 13 of pregnancy, ewes were anesthetized with sodium pentobarbital (25 mg/kg body weight) and reproductive organs exposed through a midventral abdominal incision. Pregnancy was confirmed by flushing the uterus with 20 ml of sterile saline and detection of an embryo. Corpora lutea were removed from ovaries and immediately snap frozen in liquid nitrogen and stored at -70°C for future analysis. Luteal tissue was collected from ewes on Day 4 and Day 13 of the estrous cycle in the manner described for tissue collection for Day 13 of pregnancy; however, uterine flushing was omitted from the procedure. Tissues were analyzed for PGDH enzymatic activity and mRNA encoding PGDH, COX-2, and glyceraldehyde 3-phosphate dehydrogenase (GAP3DH). Blood samples were collected before surgery and assayed for progesterone by RIA [24] to confirm the functionality of the corpus luteum.

Enzyme Assay for PGDH

Enzymatic activity of PGDH was measured in luteal homogenates utilizing a modification of a previously described assay [23]. Luteal tissue was homogenized on ice, in cold 100 mM phosphate buffer/2 mM nicotinamide adenine dinucleotide (NAD⁺; PGDH assay buffer pH 7.5), for 30 sec using a polytron tissue homogenizer on setting 4. Tissue was homogenized in a volume of 1 ml of buffer per 100 µl tissue. Homogenates were centrifuged for 5 min at 5000 x g at 4°C, and the supernatant was saved and stored at -70°C until assayed. Freezing of lung tissue at -70°C did not decrease PGDH activity after a single thaw [25]. Protein concentrations were determined in 100 ml of tissue homogenate using a commercially available kit (Coomassie Plus Protein Assay Kit; Pierce Chemical, Rockford, IL). To determine PGDH activity, 300 µl of luteal homogenate (equivalent to 30 mg of tissue) were added to a 5-ml borosilicate glass tube containing 50 ng of PGF₂-Tris salt and 700 µl of PGDH assay buffer, for a total reaction volume of 1 ml. All reagents and the tubes were prewarmed to 37°C before initiation of the assay by addition of tissue homogenate. The assay was conducted at 37°C and aliquots of 100 µl of the reaction mixture were added to 5-ml borosilicate glass tubes containing 300 µl of 200 mM formic acid to stop enzymatic activity at specified times. The pH of reactants (100 µl reaction aliquot and 300 µl of 200 mM formic acid) was approximately 2.5. Appearance of PGFM was measured at 5, 10, 15, 30, 45, 60, 90, and 180 min. To obtain time 0 PGFM levels, 30 µl tissue homogenate, 5 µl of PGF₂, and 65 µl of PGDH assay buffer were added to 300 µl of 200 mM formic acid. This approach allowed measurement of PGFM in an aliquot of tissue homogenate prior to any conversion of PGF₂ to PGFM. After terminating the enzymatic activity, 1 ml of PBS (pH 7.4) was added to each aliquot to change the pH from 2.5 to 4.5 for extraction of PGFM [26]. Samples were then extracted twice with 5 ml ethyl acetate, dried under a stream of nitrogen, and reconstituted in 1 ml of PBS/0.1% gelatin and allowed to sit overnight at 4°C. Concentrations of PGFM in the aliquots were determined using a previously validated RIA [26] in three separate assays. The average intraassay coefficient of variation was 5.9% at 20% binding and 11.8% at 80% binding, and the interassay coefficient of variation was 1.7% at 50% binding.

To assure that PGFM appearance was due to enzymatic catabolism of PGF₂ and not PGFM contamination or spontaneous conversion to PGFM, every enzyme assay contained duplicate samples containing buffer alone and PGF₂ + buffer. No PGFM was detected in any of these samples at any time. To insure that conversion of PGF₂ to PGFM proceeded to completion with no appreciable reverse reaction (PGFM to PGF₂), 100 µl of lung homogenate (10 mg tissue equivalent) was incubated with 50 ng of PGFM. There was no evidence of conversion of PGFM to PGF₂. To establish with a greater degree of certainty that conversion of PGF₂ to PGFM was a direct result of tissue-specific PGDH activity and not some other mechanism, tissues known to possess or be devoid of PGDH activity were assayed for PGDH activity.

Cloning of PGDH

A 394-base pair (bp) cDNA encoding ovine 15-hydroxyprostaglandin dehydrogenase (PGDH394) was generated utilizing reverse transcription (RT) polymerase chain reaction (PCR) (Perkin Elmer Cetus, Norwalk, CT). Polyadenylated mRNA (poly[A]⁺ RNA) was isolated from ovine lung and luteal tissue collected on Day 4 of the estrous cycle with oligodeoxythymidine (oligo[dT]) cellulose as previously described [27, 28]. Briefly, tissue was homogenized using a polytron tissue homogenizer in lysis buffer (0.2 M NaCl, 200 mM Tris HCl, pH 7.5, 1.5 mM MgCl₂, 2% SDS, 400 µg/ml proteinase K). Homogenized tissue was then incubated for 90 min at 42°C in lysis buffer, NaCl was adjusted to 0.5 M, genomic DNA was sheared by 12 passages through a 22-gauge needle, and lysates were further incubated in the presence of 5 mg oligo(dT)/100 mg tissue (Promega, Madison, WI) for 60 min at room temperature. Oligo(dT) was then washed three times with binding buffer (0.5 M NaCl, 200 mM Tris HCl, pH 7.5, 1.5 mM MgCl₂, 2% SDS) and eluted with 300 µl of elution buffer (10 mM Tris HCl, pH 7.5, 1 mM EDTA). Polyadenylated RNA was reverse transcribed using Ready-To-Go RT-PCR beads (Amersham-Pharmacia Biotech, Uppsala, Sweden) and incubated at 37°C for 1 h and 95°C for 10 min using random hexamers as primers. Upon completion of reverse transcription, PCR primers were added and PCR carried out according to the following protocol: [(95°C, 1 min; 48°C, 1 min; 72°C, 1 min; 30 cycles), (95°C, 1 min; 48°C, 1 min; 72°C, 10 min; 1 cycle)]. Primers were designed based on the sequence of Bovis bubalus (water buffalo; Genebank accession number AJ222837) PGDH, with 5'-PGDH (5'-ATGCACGTGAACGGCAAAGTG-3') and 3'-PGDH (5'-TGCCACCTTCGCCTCCATTTT-3') corresponding to bases 1–21 and 373–394, respectively. The resulting 394-bp cDNA was gel purified and ligated into pGEM-Easy (Promega), then competent Escherichia coli (DH5) cells were transformed with the resulting construct. Transformed colonies were screened by PCR for plasmids containing PGDH insert using the original PGDH primers. Plasmid containing the 394-bp PGDH cDNA was sequenced (Geneseek, Lincoln, NE). Sequence analysis (Geneworks; Intelligenetics, Upland Park, CA) led to the determination that the cDNA was 93.1%, 98.2%, 87%, and 89.3% homologous to human, water buffalo, rat, and guinea pig PGDH, respectively (data not shown).

Quantitative-Competitive RT-PCR Assay for PGDH

A quantitative-competitive (QC)-RT-PCR assay was developed to determine levels of mRNA encoding PGDH in luteal tissue. A competitor cDNA encoding a truncated version (308 bp) of the PGDH cDNA was generated by restriction digestion of the original PGDH cDNA with KasI (Promega) and BbsI (New England Biochemical, Beverly, MA). The resulting overhangs in the linearized plasmid were filled in by treatment with Klenow fragment of E. coli DNA PolI (Promega). The linearized plasmid was religated with T4 DNA ligase (Promega) and transformed into competent DH5 E. coli, then plated on ampicillin-containing (50 µg/ml) agar. Colonies were screened for the presence of a 308-bp cDNA by PCR using PGDH primers. A positive clone was identified and the presence of a partial cDNA encoding PGDH, with an internal 86-bp deletion, was confirmed by sequence analysis (Geneseek).

Plasmids containing native and competitor PGDH cDNAs were linearized with BamHI restriction endonucleases and reverse transcribed using a Maxiscript T7 kit (Ambion, Austin, TX) as per manufacturer's instructions. Standards were generated by diluting known amounts of RNA standard with the first point containing 10 fmol of PGDH RNA and subsequent points containing 0.5 dilutions for a total of nine standards. Each tube of standard or sample in the assay contained 625 amol (10^-18 moles; amol; midpoint of the standard curve) of competitor. Standards and samples were reverse transcribed for 90 min at 42°C, then cDNAs were amplified as follows: 95°C, 30 sec; 47°C, 30 sec; 72°C, 30 sec; 35 cycles, followed by 95°C, 30 sec; 47°C, 30 sec; 72°C, 10 min; one cycle. Standards and samples (1 µg poly[A]⁺ RNA) were reverse transcribed for 90 min at 42°C in a 25-µl reaction volume using random hexamers (4 µM) as primers. Reverse transcription was conducted in the following buffer: 50 mM Tris HCl, pH 8.3, 37.5 mM KCl, 0.16 mM each of dNTPs, and 1.5 mM MgCl₂. Amplification was conducted in the following buffer: 50 mM KCl, 10 mM Tris HCl, pH 9.0, 0.1% Triton X-100, 0.16 mM each of dNTPs, and 1.2 mM MgCl₂, 3.2 µM primers. The PCR products were subjected to electrophoresis on 6% polyacrylamide gels and stained with ethidium bromide. The gels were visualized on a UV transilluminator and the image captured with a charge-coupled device camera and analyzed using GelExpert software (Nucleotech, San Mateo, CA). The concentration of mRNA encoding PGDH in samples was determined by comparison to the standard curve, expressed as the ratio of the log of the intensity of the native band to the log of the intensity of the competitor band. Concentrations of mRNA encoding PGDH were expressed as amol PGDH transcript/µg poly(A)⁺ RNA.

Quantitative-Competitive RT-PCR Assay for COX-2

Plasmids containing COX-2 native and competitor cDNAs were kindly provided by Dr. Milo Wiltbank (University of Wisconsin, Madison). Briefly, standard and competitor cDNAs encoding bovine COX-2 were generated from bovine endometrial RNA utilizing RT-PCR. Primers were based on the highly homologous sequence of human, mouse, and rat COX-2 and the sequences of the PCR primers were as follows: upstream primer: 5'-AGGTGTATGTATGAGTGTAGGA-3'; and downstream primer: 5'-GTGCTGGGCAAAGAATGCAA-3'. The 484-bp DNA was cloned into pCR II vector (Invitrogen, Carlsbad, CA), and an internal competitor (378 bp) was generated by digestion with AluI restriction endonucleases and religation of the vector. The cDNAs were oriented in the reverse direction in pCR II. The native bovine COX-2 was found to be 97.5% identical in nucleotide sequence to ovine COX-2 in the corresponding region of the gene [29].

Messenger RNA encoding COX-2 was measured with a previously validated QC-RT-PCR assay [30, 31]. Plasmids containing native and competitor COX-2 cDNAs were linearized with BamHI restriction endonucleases and reverse transcribed using a Maxiscript T7 kit (Ambion) as per manufacturer's instructions. Standards were generated by diluting known amounts of RNA standard with the first point containing 240 amol of COX-2 RNA and subsequent points containing 0.5 dilutions for a total of nine standards. Each tube of standard or sample in the assay contained 15 amol (midpoint of the standard curve) of competitor. Reverse transcription and amplification were conducted identically to the PGDH assay, except cDNAs were amplified as follows: 95°C, 30 sec; 57°C, 30 sec; 72°C, 30 sec; 35 cycles, followed by 95°C, 30 sec; 57°C, 30 sec; 72°C, 10 min; one cycle. The PCR products were analyzed in a manner identical to that utilized for analysis of PGDH. Concentrations of mRNA encoding COX-2 were expressed as amol COX-2 transcript/µg poly(A)⁺ RNA.

Semiquantitative RT-PCR for GAP3DH

To ensure the integrity of poly(A)⁺ RNA used for measurement of mRNA encoding COX-2, levels of mRNA encoding GAP3DH were measured utilizing a previously described and validated semiquanitative PCR assay [30] (the native and competitor partial cDNAs encoding GAP3DH were graciously provided by Dr. Milo Wiltbank, University of Wisconsin, Madison). The competitor GAP3DH cDNA was 850 bp in length.

Briefly, a pair of primers (upstream 5'-TGTGTCCAGTATGGATTCCACCC-3' and downstream 5'-TCCACCACCCTGTTGCTGTA-3') were used to amplify the cDNA, which was reverse transcribed for COX-2 analysis. This amplification was carried out in the presence of an approximately equimolar amount of GAP3DH competitor cDNA. Amplification conditions were identical to those used for PCR of COX-2.

Progesterone Assay

Concentrations of progesterone were determined in a single RIA; intra-assay coefficient of variation was 9.2%. All ewes had concentrations of progesterone greater than 1 ng/ml of serum and were considered to have normally functioning corpora lutea.

Statistical Analyses

Enzyme activity was expressed as average rate of conversion of PGF₂ to PGFM and normalized for the protein content of the homogenates (ng PGFM/min mg protein) during the first 30 min of the enzyme assay. These data were then used for statistical analysis of PGDH activity; comparisons between days of the estrous cycle were analyzed by Fisher's F-protected least significant difference test. Mean levels of mRNA encoding PGDH and GAP3DH were also compared utilizing Fisher's F-protected least significant difference test. Two pairwise comparisons were preplanned: 1) Day 4 versus Day 13 of the estrous cycle, and 2) Day 13 of pregnancy versus Day 13 of the estrous cycle. F-statistics yielding a probability of type I error less than 0.05 were considered statistically different.

RESULTS

Enzymatic Activity of PGDH

The average rate of production of PGFM during the linear portion (Fig. 1; 0–60 min) of the PGDH assay was directly proportional to the dose of PGF₂ added (Fig. 2, first 20 min). Brain tissue, as expected, had no significant detectable PGDH activity, lung tissue had the greatest activity, and endometrium had intermediate levels of PGDH activity (Table 1). All control samples (buffer alone, buffer + PGF₂, lung pool + PGFM, and lung pool + no PGF₂ groups) were sampled at 0, 5, 90, and 180 min (or 240 min in validation assays). None of the sample or control tissues had significant endogenous PGFM at time zero (Table 1).

View larger version (20K): [in this window] [in a new window]   FIG. 1. Correlation of PGFM appearance with tissue dose at various times. A) Fifteen minutes, B) 30 min, C) 45 min, and D) 90 min. Luteal homogenates were incubated with 50 ng/ml of PGF2 in 100 mM potassium phosphate buffer/2 mM nicotinamide adenine dinucleotide (NAD+) at 37°C. The PGFM was measured at various time points by RIA. X-axis, tissue dose (mg); y-axis, PGFM production (ng). Note that PGFM production was directly proportional to the amount of tissue homogenate added. Based on these results, it was concluded that the enzyme assay was linear through 90 min

View larger version (18K): [in this window] [in a new window]   FIG. 2. Relationship between dose of PGF2 added and PGFM measured. Lung homogenates (from 50 mg of tissue) were incubated with varying concentrations (5, 10, 50, or 150 ng) of PGF2 in 100 mM potassium phosphate buffer/2 mM nicotinamide adenine dinucleotide (NAD+) at 37°C. The PGFM was measured at various time points (n = 2 per time point) by RIA. Note that PGFM production was directly proportional to the quantity of PGF2 added

To insure that measurements of PGDH activity were made when substrate was not limiting enzymatic rate, a dose response experiment was conducted (using increasing doses of tissue homogenate) to determine the times during which the assay was linear. The average rate of conversion of PGF₂ was directly proportional to the amount of tissue homogenate added (Fig. 1), and PGFM production was linear with respect to the time of incubation (Fig. 3; 15 min, r² = 0.87, 30 min, r² = 0.93, 45 min, r² = 0.94, 90 min, r² = 0.99). Enzymatic activity of PGDH was greater in corpora lutea on both Day 4 of the estrous cycle (P = 0.03) and Day 13 of pregnancy (P = 0.05) than on Day 13 of the estrous cycle (Fig. 4).

View larger version (18K): [in this window] [in a new window]   FIG. 3. Luteal homogenates were incubated with 50 ng/ml of PGF2 in 100 mM potassium phosphate buffer/2 mM nicotinamide adenine dinucleotide (NAD+) at 37°C. The PGFM was measured at various time points (n = 3 per time point) by RIA. Note that PGFM production was directly proportional to the amount of tissue homogenate added. Note: missing point (CL, 10 mg, 10 min) was due to lost samples from that time point

View larger version (15K): [in this window] [in a new window]   FIG. 4. Rates of PGFM production by corpora lutea on Days 4 (n = 11) and 13 postovulation in nonpregnant ewes (n = 7) and Day 13 in pregnant ewes (n = 10). Average rates of PGFM production (ng PGFM/mg protein/min) were determined for individual animals for the first 30 min of the enzyme assay, and statistical analyses were performed on these data. Error bars indicate SEM. Bars with different letters vary (P < 0.05)

Messenger RNA Encoding PGDH

Levels of mRNA encoding PGDH were greater in luteal tissue of ewes on Day 4 of the estrous cycle and on Day 13 of pregnancy than on Day 13 of the estrous cycle (P < 0.001; 301 ± 47 amol/µg poly(A)⁺ RNA and 177 ± 19 amol/µg poly(A)⁺ RNA vs. 17 ± 5 amol/µg poly(A)⁺ RNA; Fig. 5). Levels of mRNA encoding PGDH were also greater in luteal tissue of ewes on Day 4 of the estrous cycle than on Day 13 pregnancy (P < 0.05).

View larger version (12K): [in this window] [in a new window]   FIG. 5. Levels of mRNA encoding PGDH in the ovine corpus luteum on Days 4 (n = 11) and 13 of the estrous cycle (n = 7) and Day 13 of pregnancy (n = 10). The PGDH levels are expressed in amol mRNA/µg poly(A)+ RNA. Error bars represent SEM. Values with a different letter are different (P < 0.01)

Messenger RNA Encoding COX-2

Figure 6 shows a typical standard curve generated by QC-RT-PCR for COX-2. Levels of mRNA encoding COX-2 were 43 ± 27 amol/µg poly(A)⁺ RNA in luteal tissue of ewes on Day 4 of the estrous cycle (Fig. 7). Levels of mRNA encoding COX-2 were 10 ± 4 amol/µg poly(A)⁺ RNA in luteal tissue of ewes on Day 13 pregnancy and were undetectable in luteal tissue of ewes on Day 13 of the estrous cycle (<1.8 amol/µg poly(A)⁺ RNA; Fig. 7).

View larger version (25K): [in this window] [in a new window]   FIG. 6. Typical QC-RT-PCR standard curve. Left: Photograph of a typical standard curve using 240 amol and 0.5 dilution for each subsequent standard, and 15 amol competitor. Right: Plot of data from the standard curve on the left

View larger version (18K): [in this window] [in a new window]   FIG. 7. Levels of mRNA encoding COX-2 and GAP3DH in the ovine corpus luteum on Days 4 (n = 11) and 13 of the estrous cycle (n = 7) and Day 13 of pregnancy (n = 13). The COX-2 levels are expressed in amol mRNA/µg poly(A)+ RNA; GAP3DH levels are expressed relative to mean levels in Day 4 corpora lutea. Levels of mRNA encoding GAP3DH did not differ across experimental groups (P = 0.77). Error bars represent SEM. *Error bars for COX-2 mRNA represent SD

The inability to measure COX-2 in corpora lutea of ewes on Day 13 of the estrous cycle was not due to degraded RNA. To confirm this, mRNA encoding GAP3DH, a constitutively expressed, unregulated gene in the corpus luteum, was measured by semiquanitative RT-PCR [30]. Messenger RNA encoding GAP3DH was detectable in all samples assayed for COX-2, and expression of GAP3DH was not different across treatments (P = 0.77; Fig. 7).

DISCUSSION

This study provides direct evidence that the corpus luteum can convert PGF₂ to PGFM. This may explain, to some extent, the mechanism of luteal resistance to PGF₂ during early pregnancy and early in the estrous cycle. Catabolism of PGF₂ locally in the corpus luteum might prevent PGF₂, of uterine origin, from reaching receptors on large luteal cells. However, because PGDH is a cytosolic enzyme, it is more likely that PGDH acts to oppose the biosynthetic function of COX-2 in the corpus luteum, preventing local accumulation of PGF₂. For example, COX-2 is localized primarily to the large luteal cells [32], so the presence of PGDH in the cytosol of large luteal cells would reduce the release of PGF₂ by this cell type.

Conversion of PGF₂ to PGFM in ovine luteal tissue is most likely due to the activity of type I PGDH and not carbonyl reductase. The presence of carbonyl reductase would allow conversion of 13,14-dihydro-15-keto PGF₂ to 9-keto-13,14-dihydro-15-keto PG (13,14-dihydro-15-keto PGE₂). If this had occurred, the complete conversion of PGF₂ to PGFM observed in our studies would not have occurred. The RIA used to measure PGFM crossreacts with 13,14-dihydro-15-keto PGE₂ at <1% [26], therefore every 100 molecules of PGE₂ catabolized to 13,14-dihydro-15-keto PGE₂, would have resulted in the apparent measurement of less than one molecule of PGFM. However, when high concentrations of luteal homogenate and PGF₂ were used in the validation studies, addition of PGF₂ resulted in appearance of an equimolar quantity of PGFM when the reaction was allowed to proceed to completion (Fig. 2). If carbonyl reductase activity had been present, some of the PGFM would have been converted to PGEM and consequently not measured by the RIA for PGFM.

There is indirect evidence that PGF₂ of luteal origin is required for luteolysis in rats. First, removal of immune cell-derived cytokines by splenectomy [33] results in delayed luteolysis. It is possible that cytokines derived from these immune cells serve a key role in stimulating luteal production of PGF₂. Indeed, cytokines such as tumor necrosis factor [34], IFN [34], and interleukin-1 [35]. This effect is reversed by injection of isolated splenocytes [36]. Furthermore, immunosuppressive doses of dexamethasone also prevent luteolysis [37]. Thus, it appears that the presence of immune cell-derived cytokines that stimulate luteal PGF₂ biosynthesis [38] are a necessary component of the luteolytic machinery.

Indeed, there is compelling evidence that a threshold dose of PGF₂ is required before the corpus luteum is committed to luteolysis. The secretion of progesterone can recover in corpora lutea exposed to a subluteolytic dose of PGF₂ [9, 39]. In ewes treated with a subluteolytic dose of PGF₂, the presence of oligonucleosomes in luteal tissue declines, while in ewes treated with a luteolytic dose, oligonucleosomes persist [39]. Thus, a major biological function of PGDH in the corpus luteum could be to prevent luteal concentrations of PGF₂ from reaching threshold levels and initiating luteolysis at an inopportune time. The fact that occurrence of elevated PGDH enzymatic activity is temporally correlated with luteal resistance to PGF₂ supports the existence of such a mechanism.

It was unexpected that the lowest levels of mRNA encoding COX-2 would be observed on Day 13 of the estrous cycle. However, because COX-2 expression is very low in the absence of stimulation by PGF₂, this might be reasonable. Corpora lutea that are sensitive to PGF₂ express COX-2 in response to PGF₂ [32]. Therefore, it seems rational that the capacity of PGF₂ to induce an increase in levels of mRNA encoding COX-2 would be associated with a corpus luteum competent to undergo luteolysis. Corpora lutea on Day 13 of the estrous cycle are responsive to PGF₂, but the important aspect of COX-2 expression might be the degree of induction and, not basal, unstimulated expression of COX-2. Expression of mRNA encoding COX-2 is upregulated by PGF₂ in the corpora lutea of ewes [22] and cows [32], while basal levels are low without stimulation in many human tissues [40]. Thus, one would expect low basal expression of an inducible gene so that the metabolic pathway would be inactive in the absence of stimulatory input. Thus, the results of this study do not indicate that the corpus luteum cannot synthesize PGF₂ on Day 13 of the estrous cycle, rather that a stimulus (PGF₂) is likely required to upregulate PGF₂ synthesis.

The fact that the levels of mRNA encoding COX-2 on Day 4 of the estrous cycle and Day 13 of pregnancy are greater than on Day 13 of the estrous cycle appears to contradict the biological model proposed. However, prior to Day 13 of the estrous cycle the corpus luteum is exposed to relatively low levels of PGF₂; thus, the relative levels of mRNA encoding COX-2 are consistent with luteal PGF₂ exposure. Thus, it is possible that pulses of PGF₂ have not reached threshold levels on Day 13 of the estrous cycle in this study, and as a result COX-2 has not been stimulated in these tissues. For example, if one compares the relative exposure of the corpus luteum on Day 13 of pregnancy to Day 13 of the estrous cycle (prior to initiation of pulsatile secretion of PGF₂ by the uterus), the corpus luteum from Day 13 of pregnancy is exposed to greater basal levels of PGF₂ [41]. Alternatively, pulses of PGF₂ have occurred on Day 13 of the estrous cycle, but the corpus luteum is slightly refractory to this stimulation with respect to COX-2 expression. The data from the current experiment are not incompatible with the theory that PGF₂ is the primary stimulus for COX-2 induction in the corpus luteum.

There is the possibility that luteal PGF₂ resistance occurs because of a blockage of PGF₂-induced expression of COX-2. This occurs on Day 4 of the estrous cycle in the cow and the ewe, the corpus luteum does not increase expression of COX-2 in response to exogenous administration of PGF₂ [32]. Because the PGF₂-resistant corpus luteum is capable of catabolizing PGF₂, it is possible that stimulatory effects of PGF₂ on luteal expression of COX-2 are blunted. This scenario is consistent with the results of the experiments reported in this manuscript.

Another possible biological role for increased steady-state concentrations of mRNA encoding COX-2 in corpora lutea resistant to PGF₂ is enhanced luteal synthesis of PGE₂ and prostacyclin (PGI₂). Regulation of PGF synthase, PGE synthase, and prostacyclin synthase could influence the type of PG produced by the corpus luteum and, consequently, alter the function of the corpus luteum. Because PGE₂ has been proposed to have antiluteolytic activity, increased expression of COX-2 and PGE synthase concurrent with decreased expression of PGF synthase, might also result in luteal resistance to PGF₂. Luteal production of prostacyclin and PGE₂ has not been investigated but merits further study.

In conclusion, the results from this experiment indicate that catabolism of PGF₂ may play a role in luteal resistance to PGF₂, while basal capacity to synthesize PGF₂ (related to level of mRNA encoding COX-2) appears to be of lesser importance.

ACKNOWLEDGMENTS

The authors extend their gratitude to M. Gallegos, D. Libsack, J. Escudero, and E.S. Blehm for their valuable technical contributions.

FOOTNOTES

First decision: 29 February 2000.

¹ This research was supported by Colorado State University, Colorado Agricultural Experiment Station (W112).

² Correspondence. FAX: 970 491 3557; kthomas{at}cvmbs.colostate.edu

Accepted: July 12, 2000.

Received: January 14, 2000.

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