Nongenomic Action of Progesterone Inhibits Oxytocin-Induced Phosphoinositide Hydrolysis and Prostaglandin F2 Secretion in the Ovin
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《内分泌学杂志》
Departments of Biochemistry/Biophysics and Animal Sciences, Oregon State University, Corvallis, Oregon 97331
Abstract
Experiments were conducted to characterize the nongenomic effects of progesterone (P4) on binding of oxytocin (OT) to its receptor and signal transduction in the ovine endometrium. The dose-response relationship of P4 to OT binding was examined. Membranes from endometrial tissue of ovariectomized hormone-treated ewes were preincubated in the presence of P4 for 1 h followed by OT receptor analysis. P4 interfered with the binding of OT in a dose-dependent manner. Endometrium was then recovered from cyclic ewes and divided into explants. Treatment consisted of two dosages of P4 and two dosages of OT. Explants were analyzed for total inositol monophosphate, bisphosphate (IP2), and trisphosphate (IP3) content. Preincubation with P4 for 10 min significantly interfered with OT stimulation of IP2 and IP3 synthesis. Oxytocin increased monophosphate production, but there was no detectable effect of P4. In the next experiment, endometrial explants were cultured in the absence or the presence of arachidonic acid. Explants were then exposed for 1 h to medium containing vehicle or P4. After incubation, explants were challenged with OT and the media were collected and analyzed for 13,14 dihydro-15-keto prostaglandin F2 by RIA. Treatment of explants with AA increased PGF2 content compared with that of controls. Brief exposure to P4 significantly decreased OT-induced PGF2 secretion from explants previously exposed to medium or AA. Collectively, these data are interpreted to indicate that the observed reduction in OT-induced IP2 and IP3 production and OT-induced PGF2 secretion was due to P4 inhibition of OT binding to its receptor.
Introduction
IT IS COMMONLY accepted that progesterone (P4) regulates the concentration of OTRs [oxytocin (OT) receptors] in the ovine uterus (1, 2). It was assumed that this was only through regulation of the OTR gene via a nuclear P4 receptor. However, Grazzini et al. (3) demonstrated that in rat uteri P4 can act nongenomically to interfere with the binding of OT to its receptor. Subsequently, Dunlap and Stormshak (4) observed the same effect of a single concentration of P4 on the binding of OT to OTR in ovine endometrial plasma membrane preparations. In this latter study, P4 was shown to bind specifically to a high affinity binding site in the plasma membrane; presumably a putative membrane P4 receptor. The reported nongenomic action of P4 in the ovine uterus is consistent with the data of Bogacki et al. (5) who reported rapid interference of OT binding by P4 in the bovine uterus. Upon stimulation by OT, the endometrial OTR (a class I G protein-coupled receptor) initiates a signal cascade resulting in the hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C- to diacylglycerol (DAG) and inositol trisphosphate (IP3) (as reviewed in Ref.6). DAG serves as a major source of arachidonic acid (AA), which upon being liberated from the metabolite of DAG by an activated phospholipase A2 (7, 8, 9), is converted to PGF2, a luteolysin in domestic ruminants (10).
Plasma membrane-associated P4 receptors (mPRs) have been identified in rat granulosa cells (11) and the bovine ovary (12). It is plausible that mPRs also exist in the ovine uterus that mediate this inhibition of OT binding; however, whether P4 inhibition is a dose-response phenomenon in the ewe has not been determined. Also, whether inhibition of OT binding by P4 is reflected by impaired signal transduction has not been ascertained. Thus, the objectives of the present research were to examine the dose-response relationship of inhibition of OT binding by P4, and to characterize the impact of this inhibition of OTR function on phosphoinositide hydrolysis and production of PGF2 in the ovine endometrium.
Materials and Methods
Animals
Mature ewes of mixed breeding were used for all experiments. For experiment one, ewes (n = 5) were ovariectomized and allowed to recover for 1 month. Ewes were then treated with 17-estradiol (E2) and P4 in a sequence to mimic an abbreviated estrous cycle. Treatment of ewes consisted of once daily injections of E2 (25 μg) sc for 2 d, followed by once daily injections of P4 (10 mg) sc for 5 d, and finally once daily injections of E2 (25 μg) sc for 3 d. Intercaruncular endometrium was collected the day after the last E2 injection (4). In experiments 2 (n = 9) and 3 (n = 5), ewes were observed for behavioral estrus twice daily by use of a vasectomized ram. After at least two estrous cycles of normal duration (16.4 ± 0.2 d) ewes were assigned to an experimental group. Ewes in both experiments were treated with 250 μg of a PGF2 analog (Estrumate; Schering-Plough, Union, NJ) im on d 14 of their estrous cycle (first day of detected estrus = d 0 of the cycle) to reduce luteal function and enhance uniformity of ewes allotted to each experiment. All experimental procedures and protocols involving ewes were reviewed and performed in accordance with the Institutional Animal Care and Use Committee guidelines at Oregon State University.
Experiment 1
Membranes were isolated from the intercaruncular endometrium of hormone-treated ovariectomized ewes (n = 5) as described by Dunlap and Stormshak (4). Endometrium (5 g) was collected from ewes under dominance of E2 to ensure an enriched population of uterine OTR. It has been demonstrated previously that the majority of uterine endometrial OT receptors are localized in the intercaruncular endometrium (13). This tissue is also more readily homogenized compared with caruncular endometrium. Endometrium was homogenized by use of a Tekmar Tissumizer (Tekmar Co., Cincinnati, OH) in 10 ml ice-cold buffer (25 mM Tris-HCl, 0.25 M sucrose, pH 7.4) with 5-sec bursts five times with a 30-sec pause between bursts. Crude homogenate was transferred to a Dounce tissue grinder (Wheaton Science Products, Millville, NJ), and 10 strokes of the pestle were used to further homogenize any remaining clumps of tissue. The homogenate was then subjected to differential centrifugation to obtain a 100,000 x g membrane preparation. Membranes were evaluated for the ability of P4 to inhibit OT binding to OTR as previously described (4). Briefly, aliquots of membranes (1 mg protein per milliliter) were adjusted to a total volume of 750 μl with a buffer of 25 mM Tris-HCl, 0.01% NaN3, and 15 mM EDTA (pH 7.4) and incubated in the presence of P4 (Steraloids, Newport, RI) concentrations ranging from 0–5 ng/ml in ethanol (final concentration of ethanol 1% vol/vol) for 1 h on a shaking platform at room temperature (25 C). After incubation, all samples were centrifuged at 100,000 x g for 1 h to pellet membranes and remove any free P4. Membranes were resuspended in 500 μl 25 mM Tris-HCl, 0.01% NaN3 buffer (to correct for loss of one third of the protein through centrifugation). Specifically bound [3H]OT to membrane receptors was determined by radioreceptor assay for OTR as described below.
Experiment 2
This experiment was conducted to determine whether P4 inhibition of OT binding would alter downstream signal transduction via phosphoinositide hydrolysis. Jugular venous blood taken from ewes immediately before surgery was centrifuged at 1130 x g, and the sera stored at –20 C until analyzed for P4 by use of RIA. Serum concentrations of P4 were measured to provide an indication of luteal function at the time of tissue collection. On d 15 of the estrous cycle, intercaruncular endometrium was collected from ewes as described above and divided into explants (42 ± 2 mg). Triplicate explants were allotted immediately after collection to a 2 x 2 factorial arrangement of treatment groups with two dosages of P4 (0 and 2.5 ng/ml; the maximal inhibitory dose as determined from experiment 1) and two dosages of OT [0 and 100 nM; saturating dose used to stimulate monophosphate (IP) production used by Mirando et al. (14)]. Explants were assayed for inositol phosphate response to OT treatment as described by Mirando et al. (14). All explants were preincubated with 10 μCi myo-[2-3H]inositol (18.5 Ci/mmol; PerkinElmer Life and Analytical Sciences, Shelton, CT) in 1 ml Krebs Ringer Bicarbonate (KRB) containing 10 μM myo-inositol (Sigma, St. Louis, MO) and 10 mM glucose for 2 h at 37 C under an atmosphere of 95% O2-5% CO2. At that time, all media were replaced with 1 ml fresh KRB and incubation was continued for 30 min as above. After 30 min, 1 ml fresh KRB containing 10 mM LiCl (to prevent dephosphorylation of inositol species) and vehicle, or LiCl and P4 were added to explants, and incubation was continued for 10 min. Vehicle or OT was then added and incubation was continued for an additional 20 min, after which time all media were removed and 1 ml 15% ice-cold trichloroacetic acid (TCA) was added to explants and then allowed to incubate on ice for 30 min. The TCA solutions were collected and extracted five times with 5 ml diethyl ether, and then neutralized by addition of 25 μl 0.5 M Tris-HCl (pH 8.0). TCA solutions were then stored at –20 C until analyzed for IP, IP2, and IP3 content by chromatographic separation as described below.
Experiment 3
Results of experiment 2 demonstrated P4 inhibition of OT-induced phosphoinositide hydrolysis. Phosphoinositide signaling serves to stimulate cellular pathways, which result in liberation of AA an immediate precursor of PGF2. Therefore, experiment 3 was conducted to determine whether P4 would impair OT-induced synthesis of PGF2. Blood samples taken from ewes immediately before surgery were processed and analyzed for P4 as described for experiment 2. On d 15 of the estrous cycle, intercaruncular endometrial tissue was collected as described above and divided into explants. Duplicate explants (109.7 ± 4.9 mg) were allotted immediately after collection to a 2 x 2 factorial arrangement of treatments, consisting of two dosages of AA [Sigma: 0 and 20 μg/ml (8)] and two dosages of P4 (0 and 2.5 ng/ml). Explants were then incubated in six-well plates on Millicell-HA inserts (0.45 μm pore size; Fisher Scientific, Pittsburgh, PA) containing Eagle’s MEM (Sigma) with additives: HEPES (25 mM), L-glutamine (0.292 g/liter), phenol red (0.011 g/liter), glucose (5 mg/ml), penicillin/streptomycin (4% vol/vol; Life Technologies, Inc., Invitrogen Corp., Carlsbad, CA), insulin (0.2 IU), MEM nonessential amino acids solution (1% vol/vol; Life Technologies, Inc.), and charcoal-treated fetal calf serum (5% vol/vol; Life Technologies, Inc.) (15). Explants were preincubated in the presence of AA or vehicle for 2 h at 37 C under a humidified atmosphere (95% O2-5% CO2). Medium was replaced after preincubation with fresh medium containing either P4 or vehicle, and explants were then incubated for an additional 60 min at 37 C. After incubation, explants were challenged with 5 μM OT (15) and incubated for 60 min at 37 C. One set of explants from each ewe was not subjected to any treatments, but was carried through the experiment exposed only to vehicle to establish basal amounts of PGF2 secretion. Media were collected and stored at –20 C until analyzed for 13,14 dihydro-15-keto prostaglandin F2 (PGFM), the inactive stable metabolite of PGF2, by RIA as described below. Basal values were subtracted from all PGFM values of treated explants to establish secretion of PGF2 as a response to OT challenge.
RIAs
P4 RIA.
Serum samples (100 μl in duplicate) of ewes from experiments 2 and 3 were extracted with benzene:hexane (1:2) as described by Koligian and Stormshak (16). To correct for procedural loss due to extraction, 11,000 dpm [1,2,6,7-3H]P4 (102.1 Ci/mmol; PerkinElmer Life and Analytical Sciences) were added to a third tube containing an aliquot of the sample. The mean extraction efficiency for samples of experiment 2 was 92.3%, and the mean extraction efficiency for samples of experiment 3 was 92.0%. RIA was performed by use of no. 337 anti-P4-11-BSA (G. D. Niswender, Colorado State University, Fort Collins, CO) at a final dilution of 1:1200. Experiment 2 samples were analyzed in two assays, and experiment 3 samples were analyzed in a single assay. The sensitivity of the P4 RIA was 0.1 ng/ml. Intra and interassay coefficients of variation for experiment 2 were 4.4 and 6.9%, respectively, and the intraassay coefficient of variation for experiment 3 was 5.5%.
Prostaglandin metabolite RIA.
The PGFM RIA was used as previously validated in our laboratory (17), and modified for media as follows. Prostaglandin-free medium (200 μl) was added to all standards in place of prosatglandin-free plasma. Media from experiment 3 explants (10-μl aliquots) were analyzed for PGFM content in triplicate. The total volume of each aliquot analyzed was adjusted to 100 μl by addition of buffer (0.05 M Tris-HCl) before being subjected to RIA using 6000 dpm [3H]PGFM (174 Ci/mmol; Amersham Biosciences, Little Chalfont, Buckinghamshire, UK). All samples were corrected for this latter dilution upon final data analysis. All data are presented as picograms of PGFM per 100 μl per milligram of tissue. RIA was performed by use of antirabbit #J57 PGFM (W. W. Thatcher, University of Florida, Gainesville, FL) and was used at a final dilution of 1:317. The sensitivity of the assay was 2.5 pg/100 μl. Intra- and interassay coefficients of variation were 5.3 and 7.9%, respectively.
Radioreceptor assay
Ovine OTR binding was determined by use of a modified procedure of Hazzard and Stormshak (17) adapted from the original methods of Mirando et al. (18). Previous research by Dunlap and Stormshak (4) revealed that endometrial plasma membranes enriched with OTR from hormone-treated ovariectomized ewes are saturated at an OT concentration of 5 nM. Preincubated membranes from experiment 1 were exposed to 5 nM [tyrosyl-2,6-3H]OT (32.5 Ci/mmol; PerkinElmer Life and Analytical Sciences) to estimate total binding, or labeled OT plus a 200-fold excess of unlabeled OT (Calbiochem, EMD Biosciences Inc., La Jolla, CA) to quantify nonspecific binding. The dose-response relationship of P4 to OT binding was evaluated by examining changes in specifically bound OT, which was calculated as the difference between total and nonspecifically bound nonapeptide.
Inositol phosphate analysis
Inositol phosphate species were separated out of TCA solutions from experiment 2 ewes by a procedure originally reported by Mirando et al. (14). Briefly, TCA solutions (1 ml) were added to Poly-Prep columns (Bio-Rad Laboratories Inc., Hercules, CA) containing 0.5 ml bed volume of analytical grade type 1-X8 anion-exchange resin (Formate Form, 200–400 mesh; Bio-Rad Laboratories Inc.), sample vials were rinsed with 0.2 ml deionized water, and rinses were applied to columns. Columns were eluted with 9 ml of deionized water (fraction 1), 2.5 ml 25 mM Na2B4O7/60 mM Na formate (fraction 2), 5 ml 0.1 M formic acid/0.2 M NH4 formate (fraction 3), 5 ml 0.1 M formic acid/0.4 M NH4 formate (fraction 4), and 5 ml 0.1 M formic acid/1.0 M NH4 formate (fraction 5). The first 2.5 ml of fractions 3, 4, and 5 were collected and analyzed for incorporation of [3H]inositol into IP, IP2, and IP3, respectively, by liquid scintillation spectrometry. Incorporations of [3H]inositol into IP, IP2, and IP3 are expressed as disintegrations per minute [3H]IPn per gram of tissue (dry weight).
Statistics
Dose-response results were analyzed by way of polynomial regression. Data on production of inositol species were analyzed statistically as a randomized complete block design by use of a two-way ANOVA. Differences among means were determined by use of the general linear model procedure of SAS (Cary, NC), using the least squared means. All PGF2 values were coded by 10 and log10 transformed to correct for heterogeneity of variances. Data were then analyzed as a completely randomized design by use of a two-way ANOVA, and differences among means were again determined by use of the general linear model procedure of SAS.
Results
Experiment 1: binding of OT to the OTR after exposure of plasma membranes to various doses of P4 in vitro
Preincubation of plasma membrane preparations with P4 significantly inhibited OT binding to the OTR in a dose-dependent manner with greatest inhibition occurring at 2.5 ng/ml in this analysis. However, binding of OT at 5 ng P4/ml was greater than at 2.5 ng P4/ml, but still less than in the absence of P4. Polynomial regression analysis of data revealed an adjusted R2 value of 96.45%, with an overall SE of the estimate of 95.32 and a mean absolute error of 52.47 (P < 0.02; Fig. 1).
Experiments 2 and 3: impairment of OTR signaling by P4 in ovine endometrial explants
Having established an effective inhibitory dose of P4 on binding of OT to the OTR, experiments 2 and 3 were conducted to determine whether inhibition of binding by OT to the OTR had a significant effect on downstream signaling. Systemic concentration of P4 from ewes of experiment 2 at the time of tissue collection was 0.62 ± 0.08 ng/ml. By visual inspection, corpora lutea were pale, indicating that luteal regression was underway. Endometrial explants were analyzed for IP, IP2, and IP3 production upon treatment with OT and P4. Preincubation of explants with P4 for 10 min interfered with OT stimulation of IP3 (P4 x OT interaction, P = 0.025; Fig. 2) and IP2 (P4 x OT interaction, P = 0.03; Fig. 3), but not OT-induced IP production (OT main effect, P < 0.001; Fig. 4). P4 alone had no significant effect on the incorporation of [3H]inositol into IP (P = 0.69). A significant OT by P4 interaction on the incorporation of [3H]inositol into IP2 and IP3, was detected because of a differential response to P4 depending upon the presence or absence of OT. This differential response to P4 is also reflected by the differences among means.
To further investigate the downstream effects of the interaction of P4 with the OTR, an experiment was conducted to determine whether nongenomic inhibition of OTR signaling in the ovine endometrium altered the synthesis and (or) secretion of PGF2. Serum concentration of P4 in ewes of experiment 3 was 3.22 ± 0.14 ng/ml, which was greater than that of experiment 2 ewes. However, this is similar to serum concentrations of P4 reported for proestrous ewes by Webb et al. (19) and less than maximal secretion of 5.67 ± 0.94 ng/ml reported by Yuthasastrakosol et al. (20) on d 12 of the estrous cycle. Pretreatment of ovine endometrial explants with AA markedly increased OT-induced secretion of PGF2 when compared with explants pretreated with vehicle (P = 0.06; Fig. 5). OT-induced secretion of PGF2 was suppressed by exposure of explants to P4 for 60 min in vitro (P < 0.01; Fig. 5).
Discussion
The results of experiment 1 clearly demonstrate a dose-dependent relationship between the binding of OT and exposure to increasing concentrations of P4. Dunlap and Stormshak (4) demonstrated that a single dose of P4 (5 ng/ ml) significantly suppressed the binding of OT to the ovine OTR, but a dose-dependent relationship was not determined. It was previously reported by Grazzini et al. (3) that in rat uteri P4 could inhibit the binding of OT to the murine OTR in a dose-dependent manner. Previous studies using the murine OTR involved a wider range of dosages, from 0.1–1000 nM P4 (3). The dosages used here correspond to a P4 range of 2–16 nM, and more closely mimic the physiological concentrations of systemic P4 measured in ewes of our experiments. Burger et al. (21) investigated the effects of progestins on human OTR transfected into various cell lines, and determined that any effects of P4 on OT binding and OTR function were nonspecific steroid effects. However, they used a range of dosages between 10–200 μM, which is greater than dosages used in our experiments, and thus could reflect steroid overloading of membranes. This premise is supported by the data of Wenz and Barrantes (22), who tested the effects of various steroids, including P4, on lipid domains using artificial membrane bilayers. These latter investigators determined that the lower the hydrophobicity of the steroid, (hydrophobicity determined by the functional group bound to C17) the more lipid-domain-disrupting activity the steroid displayed at higher molar concentrations. P4, promegestone, pregnenolone, 11-hydroxyprogesterone, and 17-hydroxypregnenolone all posses low hydrophobicity groups attached to C17 and demonstrated domain-disrupting activity at the high dosages only (20 mol percentage of 50 μM = 10 μM P4).
Grazzini et al. (3) also demonstrated that inhibition of OT binding could be reflected in attenuation of downstream signaling events initiated by OT binding to its receptor. Specifically, they demonstrated a dose-dependent relationship between P4 concentration and inhibition of OT-induced inositol phosphate production in transfected CHO cells. Results of experiment 2 clearly demonstrate a similar response in ovine endometrial explants as shown by P4 suppression of incorporation of [3H]inositol into IP3 and IP2. In nonexcitable cells, (1,4,5)IP3 is converted by a 5-phosphatase to form (1,4)IP2, which is then acted upon by a 1-phosphatase to form (4)IP. In an alternate pathway (1,4,5)IP3 is converted by a 3-kinase to (1,3,4,6)IP4, which is dephosphorylated by a series of phosphatases into (1)IP and (3)IP. All three species of IPs are generated in response to activation of PLC; however, IP3 is the main second messenger of the PLC pathway, binding specifically to receptors on the endoplasmic reticulum and liberating [Ca2+]i stores (reviewed in Ref.23). Because the KD for phosphorylation of IP is lower than the KD for dephosphorylation, this metabolism is a rapid process, allowing the cell to quickly inactivate the signaling of (1,4,5)IP3 (23). All IP species are recycled back into free inositol and eventually into phosphatidylinositol 4,5 bisphosphate (24). Because LiCl acts to block the conversion of IP species to free inositol the apparent lack of inhibition by P4 at this step in the phosphoinositide cascade is unknown. It is conceivable that OT may have stimulated an accumulation of IP species arising from the metabolic conversions of IP3 and IP2 as described above, thus masking the inhibitory effects of P4. The marked stimulatory effect of OT on incorporation of [3H]inositol into IP was significant compared with control and P4-treated explants.
Bogacki et al. (5) demonstrated that P4 could inhibit the production of PGF2 by OT in bovine endometrial explants. Data of experiment 3 show this to be the case in the ewe, even when P4 is incubated in the presence of AA, which by itself stimulates an increase of basal PGF2 production in ovine endometrial explants (8). The dosage of AA used in our study was the same as the dosage used by Lee and Silvia (8), which induced only half as much PGF2 production as the dosage of OT used in their experiment (0.1 μM OT). Therefore, the amount of AA added in our experiment would not have been sufficient to compensate for the suppression by P4 of OT-induced conversion of free AA into PGF2. The PGF2 response is downstream of receptor activation, and technically is a product of the same phosphoinositide pathway that generates IP3 and DAG. The latter compound, 1,2 diacylglycerol, is acted upon by 1,2 diacylglycerol-lipase, which removes stearic acid, converting DAG into 2 arachidonoylmonoacylglycerol, which is then acted upon by PLA2 to generate intracellular AA. Although DAG has been reported to stimulate production of PGF2 by ovine endometrial explants, (7) it is the AA that arises from DAG that is converted into this eiconsanoid (8). Because DAG conversion to AA is an integral component of OT-induced phosphoinositide hydrolysis that results in PGF2 production it may be inferred that P4 inhibition of the secretion of this eicosanoid is due to interference with the ability of OT to bind to its receptor as demonstrated in the present studies.
The mechanism by which P4 inhibits OT binding (and signal transduction) is not known. However, receptors that mediate nongenomic actions of P4 have been described in many species to date, such as Xenopus oocytes (25), rat oocytes (26), spotted sea trout ovaries (27), bovine ovaries (12), and human spermatozoa (28). These vary from classical nuclear P4 receptors (nPR) that activate rapid signaling pathways such as the activation of the MAPK pathway by nPR in Xenopus oocytes (29), to proteins identified by antibodies, to the ligand binding domain of the nPR such as in human spermatozoa (28) and murine oocytes (26). One such protein identified in this manner was later characterized as a novel protein, RDA288, which mediates the actions of P4 in spontaneously immortalized murine granulosa cells (30). A novel G protein-coupled receptor has been characterized in sea trout ovaries that specifically binds progestins, activates MAPK, and inhibits adenylyl cyclase in transfected cells (27).
Dunlap and Stormshak (4) demonstrated the presence of a high affinity binding site for P4 in ovine endometrium with a KD of 1.2 x 10–9 M, and a BMax of 800 fmol/mg protein as determined by Scatchard analysis. Competition binding studies using promegestone (R 5020) as a ligand (KD 1.74 x 10–10 M) revealed that binding of a saturating concentration of labeled R 5020 was competitively inhibited by a 200-fold excess of unlabeled R 5020, P4, RU 486, and OT. Binding of R 5020 was not inhibited in the presence of a 200-fold excess of unlabeled E2, cortisol, testosterone, or arginine vasopressin. There is further evidence of a membrane-localized PR (an mPR or the classical nPR) mediating some of the effects of P4 in steroidogenic target tissues of the ruminant. Specific binding of P4 to bovine granulosa cell plasma membranes has been reported (12). In the hypothalamus/POA of the ewe, P4 administered concurrently with E2 inhibited the E2-induced preovulatory surge of GnRH/LH, without a net increase in cellular activation, implying that P4 acted directly in this system (31). Skinner et al. (32) observed that RU 486 (an nPR antagonist) can block the ability of P4 to inhibit this process, and suggested that these effects are mediated by the nPR.
The results of our experiments further demonstrate that nongenomic actions of P4 in the ewe have a significant impact on OTR function at the plasma membrane. Whether these responses are mediated by P4 acting directly on the ovine OTR itself, or are mediated by a putative mPR or a membrane localized nPR is still to be determined. The ovine conceptus is a source of P4 (33), which could antagonize the actions of OT at both a genomic and nongenomic level at a critical stage of early conceptus development, thus providing an effective mechanism to ensure survival of the embryo or fetus. The nongenomic action by P4 may serve as a backup mechanism to preclude overstimulation of OT-induced PGF2 secretion by the uterus in both pregnant and nonpregnant (cycling) ruminants.
Acknowledgments
We express gratitude to Dr. Mark Mirando, United States Department of Agriculture-Cooperative State Research, Education and Extension Service, for providing technical advice concerning experiments designed to study phosphoinositide hydrolysis. We are indebted to Jessica Schrunk and Reed Reeve for technical assistance, and appreciate the contributions of Paul Bailey, Rachel Burke, Danae Burke, Tess Baumgartner, Brian Kitamura, and Jesse Roth for their assistance in estrous detection.
Footnotes
This research was funded by the Oregon State University Agricultural Experiment Station.
First Published Online October 27, 2005
Abbreviations: AA, Arachidonic acid; DAG, diacylglycerol; E2, 17-estradiol; IP, inositol monophosphate; IP2, inositol bisphosphate; IP3, inositol trisphosphate; KRB, Krebs Ringer Bicarbonate; mPR, membrane-associated P4 receptor; nPR, nuclear P4 receptor; OT, oxytocin; OTR, OT receptor; P4, progesterone; PGF2, prostaglandin F2; PGFM, 13,14 dihydro-15-keto prostaglandin F2; TCA, trichloroacetic acid.
Accepted for publication October 19, 2005.
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Abstract
Experiments were conducted to characterize the nongenomic effects of progesterone (P4) on binding of oxytocin (OT) to its receptor and signal transduction in the ovine endometrium. The dose-response relationship of P4 to OT binding was examined. Membranes from endometrial tissue of ovariectomized hormone-treated ewes were preincubated in the presence of P4 for 1 h followed by OT receptor analysis. P4 interfered with the binding of OT in a dose-dependent manner. Endometrium was then recovered from cyclic ewes and divided into explants. Treatment consisted of two dosages of P4 and two dosages of OT. Explants were analyzed for total inositol monophosphate, bisphosphate (IP2), and trisphosphate (IP3) content. Preincubation with P4 for 10 min significantly interfered with OT stimulation of IP2 and IP3 synthesis. Oxytocin increased monophosphate production, but there was no detectable effect of P4. In the next experiment, endometrial explants were cultured in the absence or the presence of arachidonic acid. Explants were then exposed for 1 h to medium containing vehicle or P4. After incubation, explants were challenged with OT and the media were collected and analyzed for 13,14 dihydro-15-keto prostaglandin F2 by RIA. Treatment of explants with AA increased PGF2 content compared with that of controls. Brief exposure to P4 significantly decreased OT-induced PGF2 secretion from explants previously exposed to medium or AA. Collectively, these data are interpreted to indicate that the observed reduction in OT-induced IP2 and IP3 production and OT-induced PGF2 secretion was due to P4 inhibition of OT binding to its receptor.
Introduction
IT IS COMMONLY accepted that progesterone (P4) regulates the concentration of OTRs [oxytocin (OT) receptors] in the ovine uterus (1, 2). It was assumed that this was only through regulation of the OTR gene via a nuclear P4 receptor. However, Grazzini et al. (3) demonstrated that in rat uteri P4 can act nongenomically to interfere with the binding of OT to its receptor. Subsequently, Dunlap and Stormshak (4) observed the same effect of a single concentration of P4 on the binding of OT to OTR in ovine endometrial plasma membrane preparations. In this latter study, P4 was shown to bind specifically to a high affinity binding site in the plasma membrane; presumably a putative membrane P4 receptor. The reported nongenomic action of P4 in the ovine uterus is consistent with the data of Bogacki et al. (5) who reported rapid interference of OT binding by P4 in the bovine uterus. Upon stimulation by OT, the endometrial OTR (a class I G protein-coupled receptor) initiates a signal cascade resulting in the hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C- to diacylglycerol (DAG) and inositol trisphosphate (IP3) (as reviewed in Ref.6). DAG serves as a major source of arachidonic acid (AA), which upon being liberated from the metabolite of DAG by an activated phospholipase A2 (7, 8, 9), is converted to PGF2, a luteolysin in domestic ruminants (10).
Plasma membrane-associated P4 receptors (mPRs) have been identified in rat granulosa cells (11) and the bovine ovary (12). It is plausible that mPRs also exist in the ovine uterus that mediate this inhibition of OT binding; however, whether P4 inhibition is a dose-response phenomenon in the ewe has not been determined. Also, whether inhibition of OT binding by P4 is reflected by impaired signal transduction has not been ascertained. Thus, the objectives of the present research were to examine the dose-response relationship of inhibition of OT binding by P4, and to characterize the impact of this inhibition of OTR function on phosphoinositide hydrolysis and production of PGF2 in the ovine endometrium.
Materials and Methods
Animals
Mature ewes of mixed breeding were used for all experiments. For experiment one, ewes (n = 5) were ovariectomized and allowed to recover for 1 month. Ewes were then treated with 17-estradiol (E2) and P4 in a sequence to mimic an abbreviated estrous cycle. Treatment of ewes consisted of once daily injections of E2 (25 μg) sc for 2 d, followed by once daily injections of P4 (10 mg) sc for 5 d, and finally once daily injections of E2 (25 μg) sc for 3 d. Intercaruncular endometrium was collected the day after the last E2 injection (4). In experiments 2 (n = 9) and 3 (n = 5), ewes were observed for behavioral estrus twice daily by use of a vasectomized ram. After at least two estrous cycles of normal duration (16.4 ± 0.2 d) ewes were assigned to an experimental group. Ewes in both experiments were treated with 250 μg of a PGF2 analog (Estrumate; Schering-Plough, Union, NJ) im on d 14 of their estrous cycle (first day of detected estrus = d 0 of the cycle) to reduce luteal function and enhance uniformity of ewes allotted to each experiment. All experimental procedures and protocols involving ewes were reviewed and performed in accordance with the Institutional Animal Care and Use Committee guidelines at Oregon State University.
Experiment 1
Membranes were isolated from the intercaruncular endometrium of hormone-treated ovariectomized ewes (n = 5) as described by Dunlap and Stormshak (4). Endometrium (5 g) was collected from ewes under dominance of E2 to ensure an enriched population of uterine OTR. It has been demonstrated previously that the majority of uterine endometrial OT receptors are localized in the intercaruncular endometrium (13). This tissue is also more readily homogenized compared with caruncular endometrium. Endometrium was homogenized by use of a Tekmar Tissumizer (Tekmar Co., Cincinnati, OH) in 10 ml ice-cold buffer (25 mM Tris-HCl, 0.25 M sucrose, pH 7.4) with 5-sec bursts five times with a 30-sec pause between bursts. Crude homogenate was transferred to a Dounce tissue grinder (Wheaton Science Products, Millville, NJ), and 10 strokes of the pestle were used to further homogenize any remaining clumps of tissue. The homogenate was then subjected to differential centrifugation to obtain a 100,000 x g membrane preparation. Membranes were evaluated for the ability of P4 to inhibit OT binding to OTR as previously described (4). Briefly, aliquots of membranes (1 mg protein per milliliter) were adjusted to a total volume of 750 μl with a buffer of 25 mM Tris-HCl, 0.01% NaN3, and 15 mM EDTA (pH 7.4) and incubated in the presence of P4 (Steraloids, Newport, RI) concentrations ranging from 0–5 ng/ml in ethanol (final concentration of ethanol 1% vol/vol) for 1 h on a shaking platform at room temperature (25 C). After incubation, all samples were centrifuged at 100,000 x g for 1 h to pellet membranes and remove any free P4. Membranes were resuspended in 500 μl 25 mM Tris-HCl, 0.01% NaN3 buffer (to correct for loss of one third of the protein through centrifugation). Specifically bound [3H]OT to membrane receptors was determined by radioreceptor assay for OTR as described below.
Experiment 2
This experiment was conducted to determine whether P4 inhibition of OT binding would alter downstream signal transduction via phosphoinositide hydrolysis. Jugular venous blood taken from ewes immediately before surgery was centrifuged at 1130 x g, and the sera stored at –20 C until analyzed for P4 by use of RIA. Serum concentrations of P4 were measured to provide an indication of luteal function at the time of tissue collection. On d 15 of the estrous cycle, intercaruncular endometrium was collected from ewes as described above and divided into explants (42 ± 2 mg). Triplicate explants were allotted immediately after collection to a 2 x 2 factorial arrangement of treatment groups with two dosages of P4 (0 and 2.5 ng/ml; the maximal inhibitory dose as determined from experiment 1) and two dosages of OT [0 and 100 nM; saturating dose used to stimulate monophosphate (IP) production used by Mirando et al. (14)]. Explants were assayed for inositol phosphate response to OT treatment as described by Mirando et al. (14). All explants were preincubated with 10 μCi myo-[2-3H]inositol (18.5 Ci/mmol; PerkinElmer Life and Analytical Sciences, Shelton, CT) in 1 ml Krebs Ringer Bicarbonate (KRB) containing 10 μM myo-inositol (Sigma, St. Louis, MO) and 10 mM glucose for 2 h at 37 C under an atmosphere of 95% O2-5% CO2. At that time, all media were replaced with 1 ml fresh KRB and incubation was continued for 30 min as above. After 30 min, 1 ml fresh KRB containing 10 mM LiCl (to prevent dephosphorylation of inositol species) and vehicle, or LiCl and P4 were added to explants, and incubation was continued for 10 min. Vehicle or OT was then added and incubation was continued for an additional 20 min, after which time all media were removed and 1 ml 15% ice-cold trichloroacetic acid (TCA) was added to explants and then allowed to incubate on ice for 30 min. The TCA solutions were collected and extracted five times with 5 ml diethyl ether, and then neutralized by addition of 25 μl 0.5 M Tris-HCl (pH 8.0). TCA solutions were then stored at –20 C until analyzed for IP, IP2, and IP3 content by chromatographic separation as described below.
Experiment 3
Results of experiment 2 demonstrated P4 inhibition of OT-induced phosphoinositide hydrolysis. Phosphoinositide signaling serves to stimulate cellular pathways, which result in liberation of AA an immediate precursor of PGF2. Therefore, experiment 3 was conducted to determine whether P4 would impair OT-induced synthesis of PGF2. Blood samples taken from ewes immediately before surgery were processed and analyzed for P4 as described for experiment 2. On d 15 of the estrous cycle, intercaruncular endometrial tissue was collected as described above and divided into explants. Duplicate explants (109.7 ± 4.9 mg) were allotted immediately after collection to a 2 x 2 factorial arrangement of treatments, consisting of two dosages of AA [Sigma: 0 and 20 μg/ml (8)] and two dosages of P4 (0 and 2.5 ng/ml). Explants were then incubated in six-well plates on Millicell-HA inserts (0.45 μm pore size; Fisher Scientific, Pittsburgh, PA) containing Eagle’s MEM (Sigma) with additives: HEPES (25 mM), L-glutamine (0.292 g/liter), phenol red (0.011 g/liter), glucose (5 mg/ml), penicillin/streptomycin (4% vol/vol; Life Technologies, Inc., Invitrogen Corp., Carlsbad, CA), insulin (0.2 IU), MEM nonessential amino acids solution (1% vol/vol; Life Technologies, Inc.), and charcoal-treated fetal calf serum (5% vol/vol; Life Technologies, Inc.) (15). Explants were preincubated in the presence of AA or vehicle for 2 h at 37 C under a humidified atmosphere (95% O2-5% CO2). Medium was replaced after preincubation with fresh medium containing either P4 or vehicle, and explants were then incubated for an additional 60 min at 37 C. After incubation, explants were challenged with 5 μM OT (15) and incubated for 60 min at 37 C. One set of explants from each ewe was not subjected to any treatments, but was carried through the experiment exposed only to vehicle to establish basal amounts of PGF2 secretion. Media were collected and stored at –20 C until analyzed for 13,14 dihydro-15-keto prostaglandin F2 (PGFM), the inactive stable metabolite of PGF2, by RIA as described below. Basal values were subtracted from all PGFM values of treated explants to establish secretion of PGF2 as a response to OT challenge.
RIAs
P4 RIA.
Serum samples (100 μl in duplicate) of ewes from experiments 2 and 3 were extracted with benzene:hexane (1:2) as described by Koligian and Stormshak (16). To correct for procedural loss due to extraction, 11,000 dpm [1,2,6,7-3H]P4 (102.1 Ci/mmol; PerkinElmer Life and Analytical Sciences) were added to a third tube containing an aliquot of the sample. The mean extraction efficiency for samples of experiment 2 was 92.3%, and the mean extraction efficiency for samples of experiment 3 was 92.0%. RIA was performed by use of no. 337 anti-P4-11-BSA (G. D. Niswender, Colorado State University, Fort Collins, CO) at a final dilution of 1:1200. Experiment 2 samples were analyzed in two assays, and experiment 3 samples were analyzed in a single assay. The sensitivity of the P4 RIA was 0.1 ng/ml. Intra and interassay coefficients of variation for experiment 2 were 4.4 and 6.9%, respectively, and the intraassay coefficient of variation for experiment 3 was 5.5%.
Prostaglandin metabolite RIA.
The PGFM RIA was used as previously validated in our laboratory (17), and modified for media as follows. Prostaglandin-free medium (200 μl) was added to all standards in place of prosatglandin-free plasma. Media from experiment 3 explants (10-μl aliquots) were analyzed for PGFM content in triplicate. The total volume of each aliquot analyzed was adjusted to 100 μl by addition of buffer (0.05 M Tris-HCl) before being subjected to RIA using 6000 dpm [3H]PGFM (174 Ci/mmol; Amersham Biosciences, Little Chalfont, Buckinghamshire, UK). All samples were corrected for this latter dilution upon final data analysis. All data are presented as picograms of PGFM per 100 μl per milligram of tissue. RIA was performed by use of antirabbit #J57 PGFM (W. W. Thatcher, University of Florida, Gainesville, FL) and was used at a final dilution of 1:317. The sensitivity of the assay was 2.5 pg/100 μl. Intra- and interassay coefficients of variation were 5.3 and 7.9%, respectively.
Radioreceptor assay
Ovine OTR binding was determined by use of a modified procedure of Hazzard and Stormshak (17) adapted from the original methods of Mirando et al. (18). Previous research by Dunlap and Stormshak (4) revealed that endometrial plasma membranes enriched with OTR from hormone-treated ovariectomized ewes are saturated at an OT concentration of 5 nM. Preincubated membranes from experiment 1 were exposed to 5 nM [tyrosyl-2,6-3H]OT (32.5 Ci/mmol; PerkinElmer Life and Analytical Sciences) to estimate total binding, or labeled OT plus a 200-fold excess of unlabeled OT (Calbiochem, EMD Biosciences Inc., La Jolla, CA) to quantify nonspecific binding. The dose-response relationship of P4 to OT binding was evaluated by examining changes in specifically bound OT, which was calculated as the difference between total and nonspecifically bound nonapeptide.
Inositol phosphate analysis
Inositol phosphate species were separated out of TCA solutions from experiment 2 ewes by a procedure originally reported by Mirando et al. (14). Briefly, TCA solutions (1 ml) were added to Poly-Prep columns (Bio-Rad Laboratories Inc., Hercules, CA) containing 0.5 ml bed volume of analytical grade type 1-X8 anion-exchange resin (Formate Form, 200–400 mesh; Bio-Rad Laboratories Inc.), sample vials were rinsed with 0.2 ml deionized water, and rinses were applied to columns. Columns were eluted with 9 ml of deionized water (fraction 1), 2.5 ml 25 mM Na2B4O7/60 mM Na formate (fraction 2), 5 ml 0.1 M formic acid/0.2 M NH4 formate (fraction 3), 5 ml 0.1 M formic acid/0.4 M NH4 formate (fraction 4), and 5 ml 0.1 M formic acid/1.0 M NH4 formate (fraction 5). The first 2.5 ml of fractions 3, 4, and 5 were collected and analyzed for incorporation of [3H]inositol into IP, IP2, and IP3, respectively, by liquid scintillation spectrometry. Incorporations of [3H]inositol into IP, IP2, and IP3 are expressed as disintegrations per minute [3H]IPn per gram of tissue (dry weight).
Statistics
Dose-response results were analyzed by way of polynomial regression. Data on production of inositol species were analyzed statistically as a randomized complete block design by use of a two-way ANOVA. Differences among means were determined by use of the general linear model procedure of SAS (Cary, NC), using the least squared means. All PGF2 values were coded by 10 and log10 transformed to correct for heterogeneity of variances. Data were then analyzed as a completely randomized design by use of a two-way ANOVA, and differences among means were again determined by use of the general linear model procedure of SAS.
Results
Experiment 1: binding of OT to the OTR after exposure of plasma membranes to various doses of P4 in vitro
Preincubation of plasma membrane preparations with P4 significantly inhibited OT binding to the OTR in a dose-dependent manner with greatest inhibition occurring at 2.5 ng/ml in this analysis. However, binding of OT at 5 ng P4/ml was greater than at 2.5 ng P4/ml, but still less than in the absence of P4. Polynomial regression analysis of data revealed an adjusted R2 value of 96.45%, with an overall SE of the estimate of 95.32 and a mean absolute error of 52.47 (P < 0.02; Fig. 1).
Experiments 2 and 3: impairment of OTR signaling by P4 in ovine endometrial explants
Having established an effective inhibitory dose of P4 on binding of OT to the OTR, experiments 2 and 3 were conducted to determine whether inhibition of binding by OT to the OTR had a significant effect on downstream signaling. Systemic concentration of P4 from ewes of experiment 2 at the time of tissue collection was 0.62 ± 0.08 ng/ml. By visual inspection, corpora lutea were pale, indicating that luteal regression was underway. Endometrial explants were analyzed for IP, IP2, and IP3 production upon treatment with OT and P4. Preincubation of explants with P4 for 10 min interfered with OT stimulation of IP3 (P4 x OT interaction, P = 0.025; Fig. 2) and IP2 (P4 x OT interaction, P = 0.03; Fig. 3), but not OT-induced IP production (OT main effect, P < 0.001; Fig. 4). P4 alone had no significant effect on the incorporation of [3H]inositol into IP (P = 0.69). A significant OT by P4 interaction on the incorporation of [3H]inositol into IP2 and IP3, was detected because of a differential response to P4 depending upon the presence or absence of OT. This differential response to P4 is also reflected by the differences among means.
To further investigate the downstream effects of the interaction of P4 with the OTR, an experiment was conducted to determine whether nongenomic inhibition of OTR signaling in the ovine endometrium altered the synthesis and (or) secretion of PGF2. Serum concentration of P4 in ewes of experiment 3 was 3.22 ± 0.14 ng/ml, which was greater than that of experiment 2 ewes. However, this is similar to serum concentrations of P4 reported for proestrous ewes by Webb et al. (19) and less than maximal secretion of 5.67 ± 0.94 ng/ml reported by Yuthasastrakosol et al. (20) on d 12 of the estrous cycle. Pretreatment of ovine endometrial explants with AA markedly increased OT-induced secretion of PGF2 when compared with explants pretreated with vehicle (P = 0.06; Fig. 5). OT-induced secretion of PGF2 was suppressed by exposure of explants to P4 for 60 min in vitro (P < 0.01; Fig. 5).
Discussion
The results of experiment 1 clearly demonstrate a dose-dependent relationship between the binding of OT and exposure to increasing concentrations of P4. Dunlap and Stormshak (4) demonstrated that a single dose of P4 (5 ng/ ml) significantly suppressed the binding of OT to the ovine OTR, but a dose-dependent relationship was not determined. It was previously reported by Grazzini et al. (3) that in rat uteri P4 could inhibit the binding of OT to the murine OTR in a dose-dependent manner. Previous studies using the murine OTR involved a wider range of dosages, from 0.1–1000 nM P4 (3). The dosages used here correspond to a P4 range of 2–16 nM, and more closely mimic the physiological concentrations of systemic P4 measured in ewes of our experiments. Burger et al. (21) investigated the effects of progestins on human OTR transfected into various cell lines, and determined that any effects of P4 on OT binding and OTR function were nonspecific steroid effects. However, they used a range of dosages between 10–200 μM, which is greater than dosages used in our experiments, and thus could reflect steroid overloading of membranes. This premise is supported by the data of Wenz and Barrantes (22), who tested the effects of various steroids, including P4, on lipid domains using artificial membrane bilayers. These latter investigators determined that the lower the hydrophobicity of the steroid, (hydrophobicity determined by the functional group bound to C17) the more lipid-domain-disrupting activity the steroid displayed at higher molar concentrations. P4, promegestone, pregnenolone, 11-hydroxyprogesterone, and 17-hydroxypregnenolone all posses low hydrophobicity groups attached to C17 and demonstrated domain-disrupting activity at the high dosages only (20 mol percentage of 50 μM = 10 μM P4).
Grazzini et al. (3) also demonstrated that inhibition of OT binding could be reflected in attenuation of downstream signaling events initiated by OT binding to its receptor. Specifically, they demonstrated a dose-dependent relationship between P4 concentration and inhibition of OT-induced inositol phosphate production in transfected CHO cells. Results of experiment 2 clearly demonstrate a similar response in ovine endometrial explants as shown by P4 suppression of incorporation of [3H]inositol into IP3 and IP2. In nonexcitable cells, (1,4,5)IP3 is converted by a 5-phosphatase to form (1,4)IP2, which is then acted upon by a 1-phosphatase to form (4)IP. In an alternate pathway (1,4,5)IP3 is converted by a 3-kinase to (1,3,4,6)IP4, which is dephosphorylated by a series of phosphatases into (1)IP and (3)IP. All three species of IPs are generated in response to activation of PLC; however, IP3 is the main second messenger of the PLC pathway, binding specifically to receptors on the endoplasmic reticulum and liberating [Ca2+]i stores (reviewed in Ref.23). Because the KD for phosphorylation of IP is lower than the KD for dephosphorylation, this metabolism is a rapid process, allowing the cell to quickly inactivate the signaling of (1,4,5)IP3 (23). All IP species are recycled back into free inositol and eventually into phosphatidylinositol 4,5 bisphosphate (24). Because LiCl acts to block the conversion of IP species to free inositol the apparent lack of inhibition by P4 at this step in the phosphoinositide cascade is unknown. It is conceivable that OT may have stimulated an accumulation of IP species arising from the metabolic conversions of IP3 and IP2 as described above, thus masking the inhibitory effects of P4. The marked stimulatory effect of OT on incorporation of [3H]inositol into IP was significant compared with control and P4-treated explants.
Bogacki et al. (5) demonstrated that P4 could inhibit the production of PGF2 by OT in bovine endometrial explants. Data of experiment 3 show this to be the case in the ewe, even when P4 is incubated in the presence of AA, which by itself stimulates an increase of basal PGF2 production in ovine endometrial explants (8). The dosage of AA used in our study was the same as the dosage used by Lee and Silvia (8), which induced only half as much PGF2 production as the dosage of OT used in their experiment (0.1 μM OT). Therefore, the amount of AA added in our experiment would not have been sufficient to compensate for the suppression by P4 of OT-induced conversion of free AA into PGF2. The PGF2 response is downstream of receptor activation, and technically is a product of the same phosphoinositide pathway that generates IP3 and DAG. The latter compound, 1,2 diacylglycerol, is acted upon by 1,2 diacylglycerol-lipase, which removes stearic acid, converting DAG into 2 arachidonoylmonoacylglycerol, which is then acted upon by PLA2 to generate intracellular AA. Although DAG has been reported to stimulate production of PGF2 by ovine endometrial explants, (7) it is the AA that arises from DAG that is converted into this eiconsanoid (8). Because DAG conversion to AA is an integral component of OT-induced phosphoinositide hydrolysis that results in PGF2 production it may be inferred that P4 inhibition of the secretion of this eicosanoid is due to interference with the ability of OT to bind to its receptor as demonstrated in the present studies.
The mechanism by which P4 inhibits OT binding (and signal transduction) is not known. However, receptors that mediate nongenomic actions of P4 have been described in many species to date, such as Xenopus oocytes (25), rat oocytes (26), spotted sea trout ovaries (27), bovine ovaries (12), and human spermatozoa (28). These vary from classical nuclear P4 receptors (nPR) that activate rapid signaling pathways such as the activation of the MAPK pathway by nPR in Xenopus oocytes (29), to proteins identified by antibodies, to the ligand binding domain of the nPR such as in human spermatozoa (28) and murine oocytes (26). One such protein identified in this manner was later characterized as a novel protein, RDA288, which mediates the actions of P4 in spontaneously immortalized murine granulosa cells (30). A novel G protein-coupled receptor has been characterized in sea trout ovaries that specifically binds progestins, activates MAPK, and inhibits adenylyl cyclase in transfected cells (27).
Dunlap and Stormshak (4) demonstrated the presence of a high affinity binding site for P4 in ovine endometrium with a KD of 1.2 x 10–9 M, and a BMax of 800 fmol/mg protein as determined by Scatchard analysis. Competition binding studies using promegestone (R 5020) as a ligand (KD 1.74 x 10–10 M) revealed that binding of a saturating concentration of labeled R 5020 was competitively inhibited by a 200-fold excess of unlabeled R 5020, P4, RU 486, and OT. Binding of R 5020 was not inhibited in the presence of a 200-fold excess of unlabeled E2, cortisol, testosterone, or arginine vasopressin. There is further evidence of a membrane-localized PR (an mPR or the classical nPR) mediating some of the effects of P4 in steroidogenic target tissues of the ruminant. Specific binding of P4 to bovine granulosa cell plasma membranes has been reported (12). In the hypothalamus/POA of the ewe, P4 administered concurrently with E2 inhibited the E2-induced preovulatory surge of GnRH/LH, without a net increase in cellular activation, implying that P4 acted directly in this system (31). Skinner et al. (32) observed that RU 486 (an nPR antagonist) can block the ability of P4 to inhibit this process, and suggested that these effects are mediated by the nPR.
The results of our experiments further demonstrate that nongenomic actions of P4 in the ewe have a significant impact on OTR function at the plasma membrane. Whether these responses are mediated by P4 acting directly on the ovine OTR itself, or are mediated by a putative mPR or a membrane localized nPR is still to be determined. The ovine conceptus is a source of P4 (33), which could antagonize the actions of OT at both a genomic and nongenomic level at a critical stage of early conceptus development, thus providing an effective mechanism to ensure survival of the embryo or fetus. The nongenomic action by P4 may serve as a backup mechanism to preclude overstimulation of OT-induced PGF2 secretion by the uterus in both pregnant and nonpregnant (cycling) ruminants.
Acknowledgments
We express gratitude to Dr. Mark Mirando, United States Department of Agriculture-Cooperative State Research, Education and Extension Service, for providing technical advice concerning experiments designed to study phosphoinositide hydrolysis. We are indebted to Jessica Schrunk and Reed Reeve for technical assistance, and appreciate the contributions of Paul Bailey, Rachel Burke, Danae Burke, Tess Baumgartner, Brian Kitamura, and Jesse Roth for their assistance in estrous detection.
Footnotes
This research was funded by the Oregon State University Agricultural Experiment Station.
First Published Online October 27, 2005
Abbreviations: AA, Arachidonic acid; DAG, diacylglycerol; E2, 17-estradiol; IP, inositol monophosphate; IP2, inositol bisphosphate; IP3, inositol trisphosphate; KRB, Krebs Ringer Bicarbonate; mPR, membrane-associated P4 receptor; nPR, nuclear P4 receptor; OT, oxytocin; OTR, OT receptor; P4, progesterone; PGF2, prostaglandin F2; PGFM, 13,14 dihydro-15-keto prostaglandin F2; TCA, trichloroacetic acid.
Accepted for publication October 19, 2005.
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