Components of a platelet-activating factor-signaling loop are assembled in the ovine endometrium late in the estrous cycle

O. Chami,1 G. Evans,2 and C. O'Neill1

1Human Reproduction Unit, Department of Physiology, University of Sydney, Royal North Shore Hospital of Sydney, St. Leonards, New South Wales 2065; and 2Faculty of Veterinary Science, University of Sydney, Sydney, New South Wales 2006, Australia

Submitted 11 July 2003 ; accepted in final form 10 February 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Pulsatile release of uterine prostaglandin F2{alpha} (PGF2{alpha}) induces luteolysis in ruminants. Exogenous PAF is well known to cause PGF2{alpha} release from the ovine uterus. This study examines whether the components of a PAF-signaling loop exist in sheep at the time luteolysis is normally initiated. Day 14 of the cycle was the first day the uterus responded to an infusion of PAF, inducing a significant short-term increase in circulating levels of the PGF2{alpha} metabolite. There was a significant increase of PAF concentration (P < 0.001) in the endometrium and PAF release by tissue explants (P < 0.001) from day 10 to day 16 of the cycle. Endometrial tissue PAF receptor mRNA expression was induced (P < 0.01) by estradiol and progesterone treatment of animals, and transcripts were present between days 10 and 16 of the estrous cycle. Western analysis of endometrial tissue showed marked upregulation of PAF receptor protein expression from day 14 of the cycle, and immunolocalization studies showed that the receptor expression was predominantly around the endometrial glands. PAF:acetylhydrolase was primarily located within the lumen of the endometrial glands. The study shows that a PAF-signaling loop was assembled within the ovine endometrium at the time that PGF2{alpha} pulsatility was first observed.

endometrium; luteolysis; platelet-activating factor; prostaglandin F2{alpha}; PAF:acetylhydrolase (EC 3.1.1.47)


THE PULSATILE RELEASE of prostaglandin (PG)F2{alpha} by the ovine uterus leads to the eventual demise of the ovarian corpus luteum and the termination of the estrous cycle (35). The pulsatile release of luteolytic PGF2{alpha} in ewes requires the action of the ovarian sex steroids (29). The release of PGF2{alpha} from the endometrium coincides with the pulsatile release of oxytocin from the corpus luteum and posterior pituitary (13). The oxytocin receptor is expressed in the luminal epithelium, caruncular stroma, and deep glands of the endometrium (27, 40) and is regulated by the presence of estradiol (10). Oxytocin, acting via its endometrial receptor, induces the release of PGF2{alpha} (23). Furthermore, PGF2{alpha} can act on its receptor in the corpus luteum to induce the release of oxytocin (23). It was postulated that a positive feedback loop exists between these two events (9).

Under the influence of the changing profile of sex steroids of the late luteal phase, it is proposed that the positive feedback loops lead to an escalating series of PGF2{alpha} pulses that acts to induce luteolysis. During pregnancy, the production of an embryonic interferon decreases both estrogen and oxytocin receptor expression (20, 32) and increases progesterone receptor expression (32), which has the effect of blocking luteolysis.

These observations explain many aspects of luteolytic regulation in sheep; however, uterine PGF2{alpha} pulses persist in the absence of the ovary (albeit with smaller amplitude), and luteolysis occurs without a functional pituitary (8, 19, 29). The effect of the combined removal of both the ovaries and pituitary on luteolysis is yet to be assessed; thus a redundant action of these two sources of oxytocin cannot be excluded. However, some investigators (29) have concluded that the pulsatile release of PGF2{alpha} in the absence of the ovary and luteolysis in stalk-sectioned animals may be evidence that an endogenous PGF2{alpha} pulse generator exists within the uterus.

It was shown (4) that the production and release of platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphocholine) by the ovine endometrium in ovariectomized ewes were regulated by estradiol and progesterone and that the uterus of such animals responded to PAF infusion with a pulse-like release of PGF2{alpha} measured as its stable metabolite (PGFM) (3). However, it is yet to be shown whether PAF is produced at the time of initiation of luteolysis in the normal estrous cycle or whether a PAF receptor is expressed in endometrial tissue at the time it is required to respond to PAF.

This study investigates whether the ovine endometrium during the estrous cycle expresses the components of a PAF-signaling loop and whether their expression is consistent with a role for PAF in luteolytic PGF2{alpha} release.


    MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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All experiments were performed according to the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes, and they were approved by the University of Sydney Institutional Animal Care and Ethics Committee.

Ovariectomized ewes. Merino ewes were penned indoors and exposed to a 12:12-h dark-light cycle. All ewes were anesthetized by pentobarbital sodium induction with halothane-O2-N2O maintenance and were ovariectomized with aseptic techniques. Ewes were allowed to recover for ≥6 wk before treatment began. Hormone replacement of ovariectomized ewes with progesterone and estradiol-17{beta} (Sigma Chemical, St. Louis, MO) was as previously described (2, 4, 11). This hormone regimen creates blood hormone levels similar to those achieved in the natural estrous cycle (11). In ovariectomized ewes, insertion of the estradiol implant was defined as day 1. Hormones were dissolved in vegetable oil and administered intramuscularly except luteal phase estradiol, which was delivered from Silastic implants (3 cm, 3.44 mm ID, 4.65 mm OD, SilMed, Taunton, MA) inserted subcutaneously in the foreleg on day 1.

PAF treatments. PAF (1-O-hexadecyl/octadecyl-2-acetyl-sn-glycero-3-phosphocholine, Sigma) was maintained as a stock solution in chloroform (1 mg/ml). Aliquots were evaporated to dryness under N2 in siliconized glass test tubes and brought to solution in Dulbecco's Ca2+- and Mg2+-free PBS (pH 7.4) supplemented with 2.5 mg BSA/ml (PBS-BSA, vehicle). PAF and vehicle control were sterilized by filtration through a 0.22-µm filter (Millipore, NSW, Australia), prepared as aliquants in sterile 5-ml plastic tubes (Flow Labs., NSW, Australia), and then stored at –20°C until used. PAF or vehicle was infused directly into the uterine lumen via uterine catheters (1 m, 1.4 mm OD, and 0.63 mm ID; Silastic, Critchley Products, Sydney, NSW, Australia) that were inserted surgically into each uterine horn just above the uterotubal junction site on day 8. Catheters were exteriorized through a sublumbar stab incision, stored in an ethanol-soaked plastic bag, and held in place by a body stocking.

Blood samples. When repeated blood samples were required, an indwelling catheter was placed into the jugular vein on day 8, and blood samples (5 ml) were withdrawn at the intervals indicated in each experiment and placed into heparinized tubes on ice. After centrifugation, the plasma was recovered and stored at –20°C until assayed for PGFM.

PGFM RIA. The PGF2{alpha} response to PAF was measured as the stable metabolite. Davis et al. (7) showed in sheep that >99% of the PGF2{alpha} is rapidly converted to 15-keto-13,14-dihydro PGF2{alpha} (PGFM) by the lungs after the first pass. This 15-keto derivative is stable (26). Thus PGF2{alpha} release is conventionally assessed by measurement of PGFM in peripheral blood (2, 4). The PGFM assay used was as previously described (4), and the assay sensitivity was 0.05 ng/ml. Internal quality control interassay coefficients of variance were 11.4, 9.4, and 10.6%, and intra-assay coefficients of variance were 4.5, 7.3, and 6.7%, respectively, at 0.2, 0.4, and 0.8 ng/ml.

Measurement of uterine PAF and PAF:acetylhydrolase in the cycling ewe uterus between days 10 and 16 of the cycle. Merino ewes were maintained under field conditions (University of Sydney, Camden, NSW, Australia). Estrous cycles were synchronized using 60-mg medroxy-progesterone-acetate intravaginal sponges (Repromap, Upjohn, Germany) that were inserted for 12 days. Ewes were induced to ovulate by injection of 320 IU of equine chorionic gonadotropin (Folligon; Intervet, Boxmeer, The Netherlands) intramuscularly on the day of sponge removal. Ewes expressed estrus 2 days later (day 0).

Uterine samples. Ewes were anesthetized by pentobarbital sodium induction and euthanized with an overdose of saturated KCl on days 10 (n = 3), 12 (n = 3), 14 (n = 4), or 16 (n = 4) of the luteal phase, and endometrial tissue and luminal fluids were collected. Each uterine horn was immediately removed without excess mesentery, blotted free of all blood, and placed on ice. The lumen of each was flushed with 3 ml of ice-cold sterile buffer 1 (in mM: 137 NaCl, 2.68 KCl, 11.9 NaHCO3, 1 MgCl2·6H2O, 0.41 NaH2PO4·2H2O, 0.5 glucose, and 5 HEPES, pH 7.4) just below the uterotubal junction and was collected at the cervical end. The uterine flushings were divided into 0.5-ml aliquots and stored at –20°C. The luminal fluid protein concentration was measured using a Bio-Rad Total Protein assay kit (Bio-Rad Laboratories, Hercules, CA) with BSA as a standard.

Uterine tissue treatment for measurement of PAF. Endometrial caruncles were dissected from the uterus and placed in ice-cold sterile saline (Baxter Healthcare, NSW, Australia). They were thinly sliced to avoid contamination with myometrial tissue. The slices were washed in sterile saline, and 100-mg aliquots were cultured in 1 ml of modified Eagle’s medium (Sigma) containing 3 mg of BSA/ml (CSL, Victoria, Australia) for 90 min at 37°C in 5% CO2 in air. The medium supernatant was removed and frozen at –20°C until assayed for PAF.

Samples of caruncular endometrium were homogenized for 5–6 min on ice and then sonicated (setting "1", Heat Systems W-385 sonicator, New York, NY) for 30 s and centrifuged (2,500 g, 4°C, 15 min). The protein concentration of the supernatants was measured using a Bio-Rad Total Protein assay kit. Samples were then stored at –20°C until assayed.

PAF assay. PAF was assayed by a scintillation proximity assay (SPA, Amersham, Poole, UK) according to the manufacturer's instructions (limit of detection of assay was 20 pg/ml). PAF was extracted from uterine fluids and culture medium, as described previously (4, 6). For tissue homogenates, 5 mg of protein were diluted in buffer 1 and extracted in the same manner as uterine flushings. PAF was partially purified from medium, uterine flushings, or tissue homogenates, as previously described (6), with methanol extraction followed by removal of nonpolar lipids by Sep-Pac C18 cartridges (Millipore) and then phase separation with chloroform and separation by silica thin-layer chromatography (Merck, Darmstadt, Germany) with a mobile phase of chloroform-methanol-water (65:35:6, vol/vol/vol). PAF was removed from the thin-layer chromatography plate in chloroform and reduced to dryness under nitrogen. PAF was brought to solution in assay buffer (0.05 M Tris·HCl buffer, pH 7.4, containing 0.15 M NaCl, 0.01% Triton X-100, 0.1% gelatin, and 0.18 mM thimerosal). The efficiency of extraction was assessed by monitoring the recovery of tracer [3H]PAF added to some samples. The average recovery was 72 ± 7% (SE) for samples extracted with tracer [3H]PAF.

PAF:acetylhydrolase measurement in the uterus tissue and luminal fluids. Uterine luminal fluids collected above and uterine tissue homogenates collected on days 10, 12, 14, and 16 were analyzed to assess any changes in PAF:acetylhydrolase (EC 3.1.1.47 [EC] ) activity in the uterus.

PAF:acetylhydrolase assay. Assays were performed as previously described (21), with modification (25), by measuring the release of [3H]acetate from 1-hexadecyl-2-[3H]acetyl-sn-glycero-3-phosphocholine ([3H]PAF; 262.7 GBq/mM). [3H]PAF was purchased from New England Nuclear (Boston, MA), and 1-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (unlabeled PAF) was acquired from NOVA Biochem (Laufelfingen, Switzerland). Samples were added to buffer 1 containing 5 µM [3H]PAF (5.25 GBq/mM). Reactions were stopped after 15 min at 37°C by the addition of BSA (1.7 mg/ml) to buffer 1, followed by the addition of trichloroacetic acid [final concentration of 9.3% (wt/vol)] at 4°C for 30 min on ice followed by centrifugation of the precipitate at 1,500 g for 10 min. The substrate remains associated with the BSA and is thus precipitated, whereas the product (acetate) is released into the aqueous phase and is therefore detected in the supernatant. After removal of the precipitate by centrifugation, the supernatant was mixed with 2 ml of Eco-lite scintillant (ICN Biochemicals, NSW, Australia), and the [3H]acetate was counted on a Packard Tricarb model 1500 scintillation counter (Canberra Packard, NSW, Australia).

Partial characterization of PAF:acetylhydrolase activity was also performed. Ionic requirements were determined by the presence or absence of NaCl, MgCl2, and CaCl2 or by addition of 10 mM EDTA. Treatment with deoxycholate (Sigma) assessed the role of lecithin:cholesterol acetyltransferase (EC 2.3.1.43 [EC] ). Lipoprotein association was measured by cholesterol hemisuccinate agarose (Sigma) batch chromatography, as previously described (39).

Gene expression. Relative changes in mRNA expression for the PAF receptor in uterine endometrium from cycling ewes on days 10, 12, 14, and 16 and from ovariectomized ewes on days 12 (n = 3), 13 (n = 3), 14 (n = 3), 15 (n = 3), and 16 (n = 3) were assayed by Northern analysis. Tissue (100 mg) was finely homogenized in 1 ml of TRIzol Reagent (Life Technologies, Gaithersburg, MD). RNA was precipitated with isopropyl alcohol (BDH Laboratory Supplies), washed in 75% (vol/vol) ethanol (BDH), and air-dried. Isolated RNA was treated with 2 U of RQ1 DNase (Promega, Madison, WI) and incubated at 37°C for 30 min, followed by phenol-chloroform reextraction. The RNA pellet was dissolved in double-autoclaved Milli-Q water in the presence of RNase Inhibitor (Promega; final concentration 1 U/ml).

DNA probes were prepared by PCR. Ovine blood genomic DNA was obtained with a Nucleospin Blood Kit (Macherey-Nagel, Duren, Germany). PCR was performed as previously described (5). PCR products were sequenced and labeled using Ready.To.Go DNA labeling beads (Pharmacia, Piscataway, NJ) according to the manufacturer's instructions.

The size of the PCR product and the sequences of primers were as follows: {beta}-actin 243-bp fragment, forward 5'-CGTGGGCCGCCCTAGGCACCA-3', reverse 3'-TTGGCCTTAGGGTTCAGGGGG-5' (37). Ovine lung PAF receptor (GenBank accession no. AFO99674) primer sequences used were the same as those used to detect the PAF receptor in the sheep lung (14) [374-bp fragment, forward 5'-TGCTCTTCTTGATCA-3', reverse 3'-GAAGATGTGGATGATGAGGACTGGC-5' (14); Fisher Biotech, Perth, WA, Australia].

Endometrial and lung RNA (10 µg/lane) was run on 0.8% formaldehyde agarose gels and transferred onto Zetaprobe nylon (Bio-Rad, Richmond, CA) in 10x sodium chloride-sodium citrate buffer (SSC; 3 M NaCl, 0.35 M sodium citrate). The blot was prehybridized in hybridization buffer (50% formamide, 7% SDS, 5x Denhardt's solution, and 5x SSC) for 4 h at 42°C and then probed for 16–18 h at 42°C in a shaking water bath. After being washed in graded concentrations of SSC for 15 min at room temperature (RT), membranes were exposed for 30 h on a Phosphor-Imager (Molecular Dynamics, Sunnyvale, CA).

All blots were stripped, incubated with 0.01x SCC + 0.025% SDS at 80°C for 1 h in a vigorously shaking water bath and then reexposed for 24 h on the Phosphor-Imager to confirm that the DNA was removed. The blot was reprobed (as described above) for actin as a positive control. Images were analyzed using the ImageQuant program, version 3.3 (Molecular Dynamics).

Immunolocalization studies. Endometria were snap frozen in isopentane cooled in liquid nitrogen, stored at –70°C, and subsequently cut into 4-µm sections for immunolocalization studies. Sections of endometrium, brain, and lung were fixed in acetone for 10 min at RT. Tissue was washed in PBS containing 1% BSA + 1% Tween 20, pH 7.4 (TBS; Sigma). Endogenous peroxidases were quenched using 3% vol/vol hydrogen peroxide and blocked in 5% goat serum for 6 h at RT. Nonimmune IgG was used as control and incubated under the same conditions and concentrations as the primary antibodies. The PAF receptor was detected using a mouse monoclonal antibody (Alexis, San Diego, CA) diluted 1:750 with TBS and incubated for 24 h at 4°C. Plasma PAF:acetylhydrolase was detected by a rabbit polyclonal antibody (Cayman Chemical, Ann Arbor, MI), diluted 1:1,500, and incubated for 48 h at 4°C. Sections were stained using a DAKO LSAB2 System Peroxidase Kit (Dako, Carpinteria, CA) and counterstained in Mayer's hematoxylin (Sigma).

Western blotting for the PAF receptor in uterine tissue. To measure changes in protein expression of the PAF receptor in the sheep uterus across the cycle days 10, 12, 14, and 16, uterine caruncular endometrium underwent Western blotting. The PAF receptor was detected in human uterine tissue (1) and was used as a positive control. Murine uterine endometrium from PAF receptor knockout (15), obtained from the Department of Biochemistry and Molecular Biology, University of Tokyo, and from wild-type (C57 Bl/6 background) animals, was also used as a positive control.

Ovine uterine tissue was minced with a sterile scalpel blade over ice and transferred to a sterile 1.5-ml tube. Pregnant mouse uterine horns were flushed with 150 µl of PBS to remove any residual luminal fluids. The uterine horn was opened, and the endometrium was scraped off using a blunt scalpel blade and washed into a centrifuge tube with 50 µl of ice-cold PBS solution. Endometria were homogenized on setting "1" with a Heat Systems Ultrasonic W-385 sonicator (New York, NY) for 30 s.

Protein was lysed from all uterine tissue by the addition of ice-cold 2x extraction buffer (2x PBS, 2% Triton X-100, 24 mM deoxycholic acid, 0.4 mM Na vanadate, 1% NP-40, 0.2% sodium dodecyl sulfate, 20 mM NaF, 20 mM Na4P2O7, 2 mM PMSF, 3.08 µM aprotinin, 42 µM leupeptin, and 2.91 µM pepstatin A) for 10 min at 4°C (with vortexing). Tissue was then centrifuged at 3,000 g for 10 min at 4°C, and the supernatant was removed. Protein concentrations were assessed as above. Total protein (200 ng) was diluted with 5x Laemmli buffer (50 mM Tris·HCl, 5 mM EDTA, pH 8.0, 12.5% SDS, 0.05% bromophenol blue, and 5% {beta}-mercaptoethanol), boiled for 10 min, and size separated using 12% SDS polyacrylamide gels on a Bio-Rad Mini-PROTEAN II system. Proteins were blotted into polyvinylidene difluoride membranes (Hybond-P, Amersham Pharmacia) in a semi-dry blotting apparatus overnight by use of transfer buffer (12 mM Tris, PH 7.0, 96 mM glycine, and 20% methanol). Nonspecific binding was blocked by 5% skim milk in PBS supplemented with 0.05% Tween 20 (PBST, blocking buffer) at room temperature for 1 h. Membranes were probed overnight at 4°C in 5% skim milk in PBST with PAF receptor mouse monoclonal antibody (Alexis) diluted 1:500 with blocking buffer. A second horseradish peroxidase-conjugated antibody was then applied for 1 h in RT (1:1,000 diluted goat anti-mouse antibody; Jackson ImmunoResearch Laboratories, West Grove, PA). Membranes were developed using Femto Maximum Chemiluminescent Substrates diluted 1:2 (Pierce, Rockford, IL) for 5 min at room temperature. The membranes were then exposed to an X-Ray film (CL-XPosure Film, Pierce) for various times.

Data analysis. All statistical analyses were performed using SPSS for Windows (release 11.5). For endometrial assays (including receptor expression), homogeneity of variance was confirmed by Bartlett's test, and then univariate regression analysis with the General Linear Model was used. The model included the analyte (PAF concentration, PAF:acetylhydrolase activity, PAF receptor mRNA band intensity) as the dependent variable. The effect of day of cycle was the fixed-factor main effect. In the case of PAF release from endometrial tissue in vitro, where several explant cultures from each animal were set up and assayed, assay replicate was also tested as a fixed factor, and full factorial analysis was performed. For PAF receptor mRNA expression studies, {beta}-actin expression was used as a covariate in analysis of the PAF receptor. In all cases where main effects were significant, the least significant difference test was used for post hoc comparisons of individual means. Significant differences detected are shown in Figs. 2, 3, and 5. In the case of blood sampling from ewes over a period of time after PAF treatment, repeated-measures ANOVA was used to assess the effect of day of the cycle on PAF-induced changes in serum PGFM (Fig. 1).



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Fig. 2. PAF production by endometrium. A: PAF concentration of freshly collected tissue; B: PAF released by tissue explants upon culture in vitro for 90 min. Tissue was from intact ewes on day 10 (n = 3), 12 (n = 3), 14 (n = 4), or 16 (n = 4) of cycle. Values are means + SE. Values with different superscript letters were significantly different from each other at P < 0.001 (A) and P < 0.01 (B). Values with the same superscript letters were not significantly different (P > 0.05).

 


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Fig. 3. A: PAF concentration in luminal fluids. B: PAF:acetylhydrolase activity in luminal fluids. Fluids were collected from synchronized ewes on days 10 (n = 3), 12 (n = 3), 14 (n = 4), and 16 (n = 4) of cycle. Values are means + SE. Values with different superscript letters were significantly different from each other at P < 0.05, **P < 0.01, or ***P < 0.001. Values with the same letter are not significantly different (P > 0.05).

 


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Fig. 5. Representative Northern blots and histograms for endometrial PAF receptor (PAF-R) mRNA in ovariectomized ewes administered progesterone and estradiol (A) and cycling ewes (B). Actin was used as control. Values are expressed as ratios of uterine PAF-R to uterine actin (means + SE). Values with different superscript letters were significantly different from each other at P < 0.05 (A) or P < 0.01 (B), and those with the same letter were not significantly different (P > 0.05). C: Western blotting results for PAF-R in ovine uterine cyclic caruncular endometrium, human uterine tissue (Hu), C57 PAF-R+/+ (wt) murine uterine endometrium, and PAF-R knockout murine uterine endometrium (–/–).

 


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Fig. 1. Responses of stable metabolite of prostaglandin F2{alpha} (PGFM, means + SE) to intrauterine infusion of platelet-activating factor (PAF; 200 µg/horn) on days 12 (n = 3), 13 (n = 3), 14 (n = 3), 15 (n = 3), and 16 (n = 3) or vehicle control, day 15 (n = 3) in ovariectomized ewes that received progesterone + estradiol hormone replacement. Treatments with different superscript letters are significantly different from each other: P < 0.05; **P < 0.01, by repeated-measures ANOVA.

 

    RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
PGF2{alpha} release in response to PAF. Ovariectomized ewes given estradiol and progesterone replacement to mimic the estrus cycle first showed responses to uterine infusion of PAF with a recognizable PGFM pulse on day 14 (P < 0.05; Fig. 1). The response to PAF showed a significant increase in peak amplitude on subsequent days (days 15 and 16, P < 0.05) of treatment, and on day 16 the duration of the peak was greater than on earlier days. Challenge with PBS-BSA vehicle did not elicit a PGFM response.

PAF levels and PAF:acetylhydrolase activity in the cyclic uterus changed as luteolysis progressed. There was a significant (P < 0.001) effect of day of the cycle on the amount of PAF detected in endometrial tissue. PAF was not detected in endometrial caruncular tissue on day 10 of the estrous cycle in intact ewes, but it was first detected on days 12 and 14 and showed a further significant increase on day 16 (Fig. 2A). There was also a significant effect of day of treatment (P < 0.001) on the release of PAF by endometrial explants (Fig. 2B), but no significant between-sample effect within each animal (P > 0.05). The amount of PAF released showed a significant increase (P < 0.01) on each successive day of sampling between days 10 and 16. The increased amount of PAF released by the endometrium in vitro late in the cycle contrasted with the amount of PAF detected within the uterine lumen (Fig. 3A). There was a significant quadratic effect of day of the cycle (P < 0.001) on the PAF concentration within luminal fluids. The PAF concentration decreased from day 10 to day 12 (P = 0.002) and then rebounded on day 14 to levels not significantly different from day 10 (P > 0.05) and continued to increase (P < 0.01) on day 16.

There was a highly significant (P < 0.001) overall effect of the day of cycle on the PAF:acetylhydrolase activity in the uterine luminal fluids. Although there was no significant difference between days 10 and 12 (P > 0.05), a significant increase (P < 0.01) in activity occurred on day 14 and day 16. From days 10 to 16 there was an average 22-fold increase in PAF:acetylhydrolase activity in luminal fluids (Fig. 3B).

Most of the PAF:acetylhydrolase activity from both luminal fluids (96%) and endometria (98%) bound to cholesterol hemisuccinate agarose. PAF:acetylhydrolase activity (Table 1) was unaffected by monovalent or divalent cations, and treatment with deoxycholate did not inhibit the enzyme. These results are consistent with the enzyme having the characteristics of the plasma form of PAF:acetylhydrolase (33, 38).


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Table 1. Partial characterization of PAF:acetylhydrolase activity in uterine luminal fluids and endometrial homogenates

 
The PAF receptor and PAF:acetylhydrolase are present in the endometrium during luteolysis. An immunodetectable PAF receptor was associated with the endometrial glands of day 10 and day 16 tissue in both ovariectomized hormone-replaced and cyclic uterine tissue (Fig. 4A). There was no obvious staining in the lumen of the glandular epithelium. The expression of mRNA for the PAF receptor increased (P < 0.01) in endometrial tissue from day 12 of ovariectomized animals given hormone replacement (Fig. 5A). During the estrous cycle, the transcript was higher by day 12 and did not change (P > 0.05) over the study period (Fig. 5B).



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Fig. 4. A: immunolocalization of PAF receptor protein in uterine endometrium of ovariectomized (Ovx) ewes, cyclic (Cyc) animals and negative isotype IgG control and uterine tissue (x200 magnification). B: plasma-type PAF:acetylhydrolase (x300 magnification) in ovine endometrium of cyclic animals and negative isotype IgG control.

 
Immunolocalization of the plasma-type PAF:acetylhydrolase (Fig. 4B) showed that it was primarily localized within the lumen of the glands on day 16 of the cycle, there being comparatively little staining within the stroma and no detectable staining within the glandular epithelium. There was relatively little staining within the glandular lumen on day 10. The high PAF:acetylhydrolase activity within fluids collected from the uterine lumen (Fig. 3B), together with the demonstration of its localization to the glandular lumen, indicates that the increased PAF:acetylhydrolase activity in endometrial tissue during the cycle resulted primarily from an increase in enzyme concentration within the uterine luminal spaces.

Figure 5C shows that PAF receptor protein expression was markedly increased from day 14 in the ovine caruncular endometrium. The PAF receptor was present in the human uterine tissue and in the wild-type mouse endometria, but it was not detected in endometria from PAF receptor knockout mice. This result confirms the specificity of the antibody used for the Western and immunohistochemical analyses.


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This study shows that the components of a localized loop for PAF signaling that is capable of inducing uterine PGF2{alpha} release were assembled at the expected time of onset of pulsatile release of uterine PGF2{alpha}. This evidence includes the novel observations that 1) there was initiation of PAF production and release by the endometrium in cyclic ewes at the approximate time PGF2{alpha} pulse production normally commences; 2) a PAF receptor was expressed within the endometrium, under the regulation of estradiol and progesterone, with increase in the expression of this receptor on day 14 of the cycle; and 3) that the endometrium first became capable of releasing PGF2{alpha} in response to PAF challenge as the time that luteolysis approached.

The observation of a synergistic interaction of PAF and oxytocin in inducing uterine PGFM pulses in the ewe (4) may suggest a mechanism of coordinated action between these two agents in inducing luteolysis. Thus the onset of endometrial PAF production and PAF receptor expression under the influence of estradiol and progesterone may allow the production of low-amplitude PGF2{alpha} release to commence from day 12 or day 14. It is known that low-amplitude PGF2{alpha} pulses normally precede and induce the release of oxytocin (18, 23), and it was proposed that the action of the released oxytocin on the PAF-primed endometrium might cause the substantive release of luteolytic PGF2{alpha} (4). The proposed model may incorporate the action of anti-luteolytic effects of pregnancy, because it was shown that the embryonic anti-luteolysin interferon-{tau} inhibited both PAF-induced PGFM pulses and the synergism between PAF and oxytocin (4).

There is ample evidence for PAF acting to promote uterine prostaglandin release. For instance, PAF induced an increase in PGF2{alpha} synthesis in human (30), rabbit (16), and bovine (17, 36) endometrial cells. PAF caused an increase in transcription of cyclooxygenase-2 and prostaglandin production in guinea pig (24) and bovine (17) uterine endometrial cells. However, the rapid action of PAF on the endometrium suggests that its effects may be primarily nongenomic. PAF is known to activate phospholipase A2 in several cell types (3, 17, 22), and such action in the uterus may free arachidonic acid for PG synthesis (31).

The PAF receptor detected in the endometrium is a G protein-coupled receptor that is capable of linking to a range of effectors, including phospholipase A2 (3, 17, 22) and phospholipase C (12, 34). The mobilization of polyphosphated inositols in ovine endometrium by PAF treatment is consistent with this receptor being functional in this tissue (3). The sex hormone dependency of the PAF receptor expression (Fig. 5A) and the responsiveness of the ovine endometrium to PAF are consistent with the PAF receptor promoter having two estradiol response elements (28).

Although the amount of PAF receptor mRNA did not show a significant change throughout the cycle (Fig. 5B), there was an obvious increase in protein expression as assessed by Western analysis (Fig. 5C). Immunolocalization also showed that the PAF receptor was expressed primarily by glands. The increase in protein expression may also reflect an increased number of glands seen in the uterine caruncular endometrium at this stage of the cycle.

There was substantial PAF:acetylhydrolase activity within the uterus, with its activity showing a greater than sixfold increase from day 10 to day 16 (Fig. 3B). Its presence within luminal fluids may be primarily designed to prevent bioactive PAF leaving endometrial tissue. This conclusion is supported by the observation that, whereas the PAF concentration in the endometrium and that released by explants increased from days 10 to 16 by more than sixfold and fourfold, respectively, the PAF concentration in luminal fluids increased over the same period by only ~30%. The high PAF:acetylhydrolase activity within the lumen and endometrial tissue may account for the relatively large amount of PAF that was required to be infused into the uterine lumen to induce a PGF2{alpha} response. It can be anticipated that this infused PAF would be very rapidly degraded by luminal PAF:acetylhydrolase and that the PAF concentration reaching the site of the PAF receptor-expressing cells in the stroma would be much lower.

The accumulating evidence for the presence of a sex steroid-dependent PAF-signaling loop within the ovine endometrium and its role in modulating the production of endometrial PGF2{alpha} should be considered in any description of the nature of luteolysis in the ewe.


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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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This study was supported by the Australian National Health and Medical Research Council.


    ACKNOWLEDGMENTS
 
We thank Ray Kearns and staff for care of ewes and assistance with animal surgery at the Royal North Shore Hospital. Dr. Basil Ibe (Department of Pediatrics, Harbor-UCLA Medical Center, Torrance, CA) kindly provided the ovine PAF receptor sequence before publication, and Dr. S. Ishii (Department of Biochemistry, University of Tokyo) provided PAF receptor mice. PaLMs Laboratories provided tissue-sectioning equipment and donated human uterine protein. We thank Dr. Judy Simpson, School of Public Health, University of Sydney, for assistance with statistical analysis, and Dr. Patric Delhanty and Dr. David Lu, Royal North Shore Hospital, for valuable discussions.


    FOOTNOTES
 

Address for reprint requests and other correspondence: C. O'Neill, Human Reproduction Unit, Dept. of Physiology, Univ. of Sydney, Royal North Shore Hospital of Sydney, St. Leonards, NSW 2065, Australia (E-mail: chriso{at}med.usyd.edu.au).

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.


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 DISCUSSION
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