Impact of Chronic Treatment of Ewes with Estradiol-17ß or Progesterone on Oxytocin Receptor Gene Transcription and Ovarian Oxytocin Secretion¹

^a Departments of Animal Sciences and Biochemistry/Biophysics, Oregon State University, Corvallis, Oregon 97331-6702

	ABSTRACT

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Three experiments were conducted, the objectives of which were to 1) examine the effects of exogenous estradiol (E₂) and progesterone (P₄) on uterine concentrations of oxytocin receptors (OTR) and OTR mRNA, as well as the effect of exogenous P₄ on progesterone receptors (PR) during the late luteal phase of the cycle, and 2) ascertain whether chronic E₂ treatment of ewes during the cycle would alter prostaglandin F₂ (PGF₂)-induced secretion of luteal oxytocin (OT). In experiment 1, 15 ewes were assigned to a control (n = 5; 2 ml corn oil [CO] s.c. on Days 4–14 of the estrous cycle) and two treatment groups (n = 5 each) receiving either 250 µg E₂ s.c. (Days 4–14) or 10 mg P₄ s.c. (CO on Days 4–10 and P₄ on Days 11–14). Endometria and corpora lutea were removed on Day 15 of the cycle. Mean luteal weights were greater in treated than in control ewes (p < 0.05). Endometrial concentrations of OTR and OTR mRNA were significantly greater in control than in E₂- or P₄-treated ewes. In experiment 2, five ewes each were treated s.c. with CO or 10 mg P₄ on Days 11–14 of the cycle; endometria were then removed on Day 15 for PR assay. Endometrial concentration of PR did not differ between groups. Experiment 3 consisted of 20 ewes assigned to four groups in a 2 x 2 factorial arrangement. Treatment consisted of two dosages of E₂ (0 or 250 µg/day) in 2 ml CO and two dosages of PGF₂ analogue (0 or 125 µg Estrumate). All ewes were injected s.c. with E₂ or CO for 11 days as described for experiment 1. On Day 15, all ewes received an i.v. injection of PGF₂ or saline (Time 0); then jugular blood was collected at frequent intervals for analysis of serum concentrations of OT. PGF₂ induced a release of OT in control and E₂-treated ewes (p < 0.05) compared to the value in saline-treated ewes. Collectively, these data suggest that in cycling ewes, exposure of the uterus to increased concentrations of E₂ or P₄ causes down-regulation of OTR as a consequence of suppression of the OTR gene. Chronic E₂ treatment of ewes during the cycle does not act directly on the ovary to alter the stores of luteal OT.

	INTRODUCTION

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During the past two decades, it has been hypothesized that luteolysis during the estrous cycle of ruminants occurs as a consequence of a positive feedback system between luteal oxytocin (OT), which stimulates secretion of the uterine luteolysin, prostaglandin F₂ (PGF₂), and vice versa [1–3]. An integral aspect of this feedback system is the coordinated actions of estrogen and progesterone in regulating uterine concentrations of receptor to which OT binds to initiate the pulsatile release of PGF₂.

We have previously demonstrated that chronic treatment of ewes with estradiol-17ß (E₂) beginning on Day 4 of the estrous cycle prevented an increase in endometrial concentrations of oxytocin receptors (OTR) at the expected time of luteolysis, prolonged luteal life span, and maintained normal systemic concentrations of progesterone (P₄) [4]. However, the acquired data failed to provide a clue as to whether the suppression in uterine concentrations of OTR was due to a direct effect of E₂ or was indirect via the sustained production of P₄. P₄ suppresses uterine concentrations of estrogen receptor (ER) in E₂-treated ovariectomized ewes [5] and uterine concentrations of OTR in intact [6, 7] and ovariectomized ewes [8, 9]. Thus, the sustained production of P₄ in ewes subjected to chronic E₂ treatment may account for the observed reduction in uterine OTR. Regardless of which steroid in chronic E₂-treated ewes causes the change in uterine OTR, it is not known whether the effect is genomic or nongenomic. There is at least some circumstantial evidence that E₂ may act directly on the genome to alter uterine production of OTR. In the rat, an estrogen response element (ERE) has recently been reported to exist in the 5' flanking region of the OTR gene [10]. It is conceivable that the promoter region of the ovine OTR gene also contains an ERE, thus providing for E₂ regulation of gene expression. Presently, there is no evidence for the existence of a progesterone response element (PRE) present in the promoter region of the OTR gene [10, 11]. On the other hand, there is some evidence that P₄ may be acting via a nongenomic mechanism to regulate uterine production of OT in the rat [12]. Whether the sustained concentrations of P₄ present in the chronic E₂-treated ewe alter uterine concentration of progesterone receptor (PR) during the otherwise normal period of luteolysis is of associated relevance in ascertaining whether its mode of action is genomic. Insight regarding the mechanism(s) by which P₄ in E₂-treated ewes may regulate uterine OTR might be acquired by determining whether administration of P₄ during the anticipated period of luteolysis promotes a change in uterine concentration of PR.

Ovine large luteal cells, which are the source of OT [13], have been reported to contain ERs [14], and the rat gene for OT has been shown to contain an ERE upstream of the transcription start site [12, 15]. Because chronic exposure of the ewe to E₂ results in a pharmacological state, it is conceivable that this treatment regimen may alter synthesis and (or) storage of luteal OT. Such an effect of this treatment could be ascertained by challenging the chronic E₂-treated ewe with PGF₂ to induce the secretion of luteal OT [1]. A reduction in luteal OT production may in part contribute to absence of a luteolytic mechanism in the chronic E₂-treated ewe.

The present study was conducted to 1) examine the effects of chronic E₂ and P₄ treatment on uterine concentrations of OTR and OTR mRNA, as well as the effect of exogenous P₄ on PR during the late luteal phase of the cycle, and 2) ascertain whether chronic E₂ treatment of ewes during the cycle would alter luteal OT production.

	MATERIALS AND METHODS

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Animals

Mature ewes were checked for behavioral estrus twice daily with a vasectomized ram. After at least one estrous cycle of normal duration (15.5–18.0 days), ewes were assigned randomly to treatment groups. All experimental procedures and surgeries were performed in accordance with the NIH guide for the Care and Use of Laboratory Animals at Oregon State University.

Treatments

Experiment 1. Fifteen mature ewes were assigned randomly in equal numbers to three treatment groups to compare the effects of exogenous E₂ and P₄ on uterine concentrations of OTR and OTR mRNA. Daily dosage of E₂ administered to ewes in this experiment (and experiment 3 below) was one half of that recently used in a similar series of experiments in our laboratory [4] but, nevertheless, was considered to be sufficiently pharmacological to alter uterine function as based upon the observations of Hawk and Bolt [16]. Treatments consisted of a control group and two groups receiving either 250 µg of E₂ or 10 mg of P₄ in 2 ml of corn oil (CO) per day. Injections of E₂ (s.c.) were initiated on Day 4 of the cycle and continued for a total of 11 days; P₄-treated ewes received 2 ml CO s.c. on Days 4–10 and P₄ on Days 11–14. Ewes were anesthetized and laparotomized on Day 15 as previously described [4]. All corpora lutea (CL) were counted and enucleated, and weights were recorded. An incision was made through the antimesometrial uterine tissue parallel to the long axis of the horn to expose the lumen. Approximately 3 g of caruncular and intercaruncular endometrium from the uterine horn adjacent to the ovary bearing the CL was collected, immediately frozen in liquid N₂, and stored at -80°C until assayed for OTR and OTR mRNA.

Experiment 2. This experiment was conducted to determine whether P₄ treatment of ewes, as described for experiment 1 above, altered uterine concentrations of PR. Ten mature ewes were assigned randomly in equal numbers to two treatment groups. Treatments consisted of two concentrations of P₄ (0 or 10 mg) dissolved in 2 ml of CO. Injections (s.c.) were initiated on Day 11 of the cycle and continued for a total of 4 days. All ewes were laparotomized on Day 15, and endometrial tissue was removed from the uterine horn adjacent to the ovary bearing the CL, maintained at 4°C, and immediately assayed for PR.

Experiment 3. The primary objective of this experiment was to examine the effect of chronic E₂ treatment of ewes on luteal secretion of OT in response to a PGF₂ challenge. Twenty mature ewes were assigned randomly in equal numbers to four treatment groups in a 2 x 2 factorial arrangement. Treatment consisted of two dosages of E₂ (0 or 250 µg) dissolved in 2 ml of CO and two dosages of PGF₂ analogue (Estrumate; Mobay Corp., Shawnee, KS; 0 or 125 µg). Control and E₂-treated ewes were injected s.c. for 11 days as described for experiment 1. Jugular blood samples collected by use of vacutainer tubes on Day 15 were allowed to clot overnight at 4°C before centrifugation at the same temperature for 10 min at 2450 x g. Serum was removed and stored at -20°C, but serum of only nine control and nine E₂-treated ewes was assayed for P₄ because a sample from each group was lost in storage. After collection of blood samples for P₄ analysis, all ewes received either 1 ml physiological saline or 1 ml of PGF₂ analogue (125 µg) i.v. on Day 15, with jugular blood collected in heparinized vacutainer tubes at 0 min before injection and then at 2, 5, 10, 20, and 30 min after injection. Upon collection, 10 µl of 5 mg/ml 1,10-phenanthroline (Sigma Chemical Co., St. Louis, MO) in ethanol and 20 µl 0.5 M EDTA were added to each tube to suppress oxytocinase activity. All tubes were mixed by inversion and placed on ice for transport to the laboratory, where blood was immediately centrifuged as described above. Plasma was removed and stored at -20°C until assayed for OT.

OTR Analysis

Endometrium was analyzed for OTR with a procedure described by Hazzard and Stormshak [4] according to methods adapted from Mirando et al. [17]. Single-point saturation analysis of ovine endometrium preparation assayed at 3.0 nM [³H]OT resulted in intraassay and interassay coefficients of variation of 4.3% and 14.0%, respectively.

PR Exchange Assay

Endometrial PR concentrations were analyzed using a procedure described by Slayden et al. [18] and modified as follows. Radioligand promegestone, [17-methyl-³H]-R5020 (86 Ci/mmol; Dupont NEN, Boston, MA) was diluted to a concentration of 120 nM (573 000 dpm/25 µl) in the appropriate buffers. Nuclear and cytosolic fractions were each assayed in duplicate (100 µl/tube) by addition of 25 µl of labeled R5020 (3–120 nM) or labeled R5020 plus 25 µl of 200-fold excess unlabeled R5020 for a total volume of 150 µl. To determine affinity of R5020 for glucocorticoid receptors, dexamethasone (10 nM) was added to additional samples, and duplicate assays at 8 nM concentration of [³H]R5020 were performed. There was no indication of binding of [³H]R5020 with the glucocorticoid receptor (p > 0.05, Student's t-test). Specifically bound hormone was determined by subtracting nonspecifically bound steroid (3.7 ± 0.7%) from total bound steroid. Binding of [³H]R5020 to ovine endometrium was saturable at a concentration of 8 nM of labeled ligand. Sensitivity of the assay was 16 fmol/mg DNA.

P₄ Analysis

P₄ was extracted from 100 µl of serum with benzene:hexane (1:2) and subsequently quantified by use of RIA as described by Koligian and Stormshak [19]. Analysis of a quality-control sample of P₄ (75 pg/tube) resulted in an intraassay coefficient of variation of 7.8%. Sensitivity of the assay was 0.05 ng.

OT Analysis

OT was extracted from 1 ml of plasma and assayed as described by Bertrand and Stormshak [20] using methods adapted from Abdelgadir et al. [21] and Schams [22]. For plasma extraction, a Waters vacuum manifold was utilized with Sep-Pak Plus C–18 cartridges (Waters Chromatography Division, Millipore Corp., Milford, MA). Mean extraction efficiency was 61 ± 0.5%. OT antibody was generously provided by Dr. Dieter Schams, Technical University of Munich, Freising-Weihenstephan, Germany. Analysis of a stock plasma sample of OT (3.5 pg/tube) resulted in intraassay and interassay coefficients of variation of 5.9% and 15.6%, respectively. Sensitivity of the assay was 0.125 pg.

RNA Extraction and Northern Blotting

Endometrial tissues (approximately 300 mg) from each of three control and chronic E₂-treated and P₄-treated ewes were pulverized with a mortar and pestle over a dry-ice/ethanol bath and then placed in 10 vol (3 ml) Tri Reagent (Molecular Research Center, Inc., Cincinnati, OH), a single-step total RNA isolation reagent consisting of a monophase solution of phenol and guanidine thiocyanate [23]. An equivalent quantity of mammary gland tissue from a nonlactating ewe was similarly processed to serve as a negative control. Extraction of total RNA was performed according to the manufacturer's protocol, which included procedures for homogenization of tissue, precipitation of RNA with isopropanol, and centrifugation. Final resuspension of recovered RNA was in 50 µl diethylpyrocarbonate (DEPC)-treated water, which was incubated at 55–60°C for 10 min. Recovered RNA was quantitated by A₂₆₀ spectrophotometric measurement and assessed for purity by A_260/280 ratio.

Samples were denatured at 65°C for 10 min with 4-strength sample buffer (1 part 10-strength Northern buffer [0.2 M Hepes, 10 mM EDTA, pH 7.8], 1.6 parts 37% formaldehyde, and 5 parts deionized formamide) in a ratio of 1:3. Denatured samples (30 µg total RNA) plus 2 µl of 10% bromophenol blue were loaded onto a 1.25% formaldehyde agarose gel and electrophoresed in single-strength Northern buffer for 2 h at 70 V. The gel was then rinsed for 40 min in DEPC-treated water before equilibration in 10-strength SSC buffer (single-strength SSC = 0.15 M NaCl, 15 mM sodium citrate, pH 7.0) for 20 min. Transfer of RNA onto MagnaGraph Nylon membrane (Microns Separations Inc., Westboro, MA) was performed by capillary transfer. After 12–18 h, the membrane was rinsed for 10 min in 6-strength SSC and allowed to air dry on filter paper (10–15 min), and the RNA was UV cross-linked to the membrane for 30 sec. The membrane was remoistened in single-strength SSPE (3 M NaCl, 0.2 M NaH₂PO₄·H₂0, 20 mM EDTA, pH 7.4) before staining for 3 min (0.02% methylene blue, 0.3 M sodium acetate, pH 5.5) and then destaining for 15 min in single-strength SSPE to visualize the 18S and 28S rRNA bands. After visualization and destaining, the membrane was baked for 1 h at 80°C in a vacuum oven before removal of the stain from the membrane with a strip buffer (0.1-strength SSPE/0.1% SDS) for 15 min.

Probes for OTR and 18S RNA were made as follows. The template for the OTR probe was a 0.8-kilobase (kb) BamHI/Pst I fragment excised from the human OTR gene plasmid insert (a gift from Dr. T. Kimura, Osaka University Medical School, Osaka, Japan). The probe was made from the cDNA template by random hexanucleotide priming (Prime-a-Gene Labeling System; Promega, Madison, WI) with Easytide [-³²P]dCTP (Dupont NEN) as the radioactive label. Unincorporated label was removed by passage through a Sephadex G–50 column (Quick Spin columns; Boehringer-Mannheim, Indianapolis, IN). The 18S probe was made from eukaryotic rRNA (1406R) [24] and end-labeled with [-³²P]dATP (Dupont NEN) using T4 polynucleotide kinase (Promega). Unincorporated label was removed using a Sephadex G–25 spin column (Boehringer-Mannheim).

Hybridization buffer (5-strength SSPE, 5-strength Denhardt's solution [0.1% each BSA, Ficoll, and polyvinylpyrrolidone; Sigma], 1% SDS, 100 mg/ml salmon testes DNA for hybridization [Sigma], and 50% deionized formamide) was incubated with the membrane at 42°C for at least 4 h by automated rotation in a hybridization oven (Robbins Scientific, Sunnyvale, CA). After incubation, the OTR cDNA probe was added to the hybridization solution to obtain approximately 4 x 10⁶ cpm/ml and incubated overnight at 42°C. Membranes were washed at room temperature for 15 min three times in single-strength SSPE/0.1% SDS, followed by one wash for 15 min at 50°C in strip buffer. Blots were then wrapped in plastic wrap and exposed to a storage phosphor screen (Molecular Dynamics, Sunnyvale, CA) for 4–5 days. Screens were scanned by a PhosphorImager SI and visualized with ImageQuant software (Molecular Dynamics). Before use of the 18S rRNA probe, blots were incubated at 70°C for 30 min in strip buffer to remove any OTR probe. Thereafter, the blots were prehybridized and then rehybridized overnight at room temperature and visualized as outlined above.

Statistical Analysis

Effect of E₂ treatment on the uterine concentrations of OTR was tested for significance by ANOVA using Statgraphics (STSC Inc., Rockville, MD). Because uterine concentrations of OTR in P₄-treated ewes were below the detection limits of the assay, data on these animals were not included in the statistical analysis. Effects of treatments on luteal weights were tested by analysis of covariance using number of CL as the independent variable among groups. Differences among group means were further tested for significance with use of orthogonal contrasts. Data on serum concentrations of P₄ and effects of P₄ treatment on the uterine concentrations of PR were tested for significance by t-test using Statgraphics (STSC Inc.). Plasma concentrations of OT were subjected to repeated measures analysis using the General Linear Models procedure of the Statistical Analysis System [25]. Data were considered significantly different at (p < 0.05).

	RESULTS

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Chronic treatment of ewes with E₂ from Days 4 to 14 of the estrous cycle or daily injection of P₄ during Days 11–14 of the cycle (experiment 1) reduced endometrial concentrations of OTR compared with that of controls (E₂, 14 ± 11, P₄ < 1 vs. controls, 348 ± 140 fmol/mg protein; p < 0.05). These data are supported by analysis of endometrial OTR mRNA, which demonstrated that both E₂ and P₄ treatments may inhibit transcription of the OTR gene (Fig. 1). All four differentially polyadenylated variants of mRNA (1.65, 2.5, 3.9, and 6.2 kb) for the ovine OTR gene [26] were visible in Day 15 endometria of control ewes (Fig. 1; lanes 8–10), but none were evident in endometria of steroid-treated ewes. Absence of OTR mRNA in endometria of treated ewes was not due to unequal loading of total RNA, because no differences in quantities of 18S rRNA among samples were detected. Additionally, the absence of detectable OTR mRNA in nonlactating mammary gland tissue is consistent with the data reported by Flint et al. [26] and indicates the specificity of the probe for the OTR mRNA.

View larger version (86K): [in this window] [in a new window]   FIG. 1. Northern blot of OTR mRNA from Day 15 ovine endometria. Each lane represents one ewe. Lane 1: ovine nonlactating mammary gland tissue (negative control). Lanes 2–4: animals received 10 mg P4 s.c. on Days 10–14. Lanes 5–7: animals received 250 µg E2 s.c. on Days 4–14. Lanes 8–10: animals received 2 ml CO s.c. on Days 4–14 (positive controls). The mRNA for OTR is represented by four differentially polyadenylated variants (1.65–6.20 kb). 18S rRNA was probed to determine equality of loading.

Treatment of ewes with P₄ from Days 11 to 14 (experiment 2) did not alter uterine concentrations of cytoplasmic PR compared with that of control ewes (P₄-treated, 200 ± 40 vs. controls, 180 ± 40 fmol/mg DNA; p > 0.05). Unfortunately, uterine concentrations of nuclear PR could not be quantitated because concentrations were below the level of sensitivity of the assay. Chronic treatment of ewes with 250 µg of E₂ daily during the cycle or administration of P₄ during the late luteal phase of the cycle (experiment 1) resulted in CL on Day 15 that were heavier than those of controls (E₂-treated, 394 ± 23, P₄-treated, 492 ± 67 vs. controls, 307 ± 43 mg; p < 0.05 for each treatment compared with controls). Consistent with these weights of CL, mean serum concentrations of P₄ in chronic E₂-treated ewes on Day 15 (experiment 3) were greater than in controls (1.33 ± 0.23 vs. 0.92 ± 0.37 ng/ml), but the difference was not significant statistically. However, the mean of control ewes is based upon eight animals because one ewe in this group had a serum concentration of 4.36 ng/ml, fivefold greater than the mean serum concentration of P₄ in the other animals. Because of this unusually increased serum concentration of P₄, this ewe was considered to be an outlier and the datum for this animal was not included in the statistical analysis.

Injection of PGF₂ analogue on Day 15 of the cycle (experiment 3) did not alter induced secretion of OT in chronic E₂-treated and control ewes (E₂ x PGF₂ x time interaction, p > 0.05; Fig. 2). However, compared with values in saline-injected ewes, plasma concentrations of OT were markedly increased by injection of PGF₂ into both chronic E₂-treated and vehicle-injected ewes (p < 0.05).

View larger version (27K): [in this window] [in a new window]   FIG. 2. Plasma concentrations of OT in control (CO) and E2-treated ewes on Day 15 of the cycle after i.v. injection of saline (S) or 125 µg PGF2 (Estrumate) at Time 0. The common estimate of the standard error was 15.18. Each point represents the mean concentration of OT of five animals per group.

	DISCUSSION

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Results of these research endeavors indicate that chronic treatment of ewes with E₂ from early to late in the estrous cycle prevented the normal increase in endometrial OTR gene transcription as reflected by the absence of measurable quantities of OTR mRNA on Day 15. In view of this observation, it is not surprising that analysis of the same endometrial tissue for OTR also revealed a significant reduction in quantities of this receptor in E₂-treated ewes. This latter finding is consistent with our recent data demonstrating that chronic E₂ treatment of ewes during the cycle caused down-regulation of uterine OTR without affecting uterine concentrations of ER [4]. The dosage of E₂ used in the current study, although only one half of that used in our previous experiments, was apparently sufficient to provoke similar changes in uterine concentrations of OTR. Similarly, the effectiveness of this dosage of E₂ in altering the luteolytic mechanism is supported in part by the greater weight of CL in treated compared with control ewes on Day 15. Thus, there is some indication that daily administration of 250 µg of E₂, like the dosage of 500 µg of E₂ used previously [4], did sustain luteal function; but no prediction can be advanced as to how long these CL would persist and continue to function once treatment ceased. Mean serum concentration of P₄ of E₂-treated ewes tended to be greater than that of controls, but the difference was not significant statistically. Ewes treated with 500 µg of E₂ from Days 4 to 24 postestrus exhibited a mean interestrous interval of over 50 days [4].

Because our previous studies demonstrated that chronic treatment of ewes with E₂ during the cycle prolonged luteal function, it was hypothesized that the sustained production of P₄ actually mediated the effect of chronic E₂ in down-regulating uterine OTR. This hypothesis was tested in experiment 1 through administration of P₄ to ewes during the late luteal phase of the cycle (Days 11–14). The effect of P₄ on endometrial concentrations of OTR and OTR mRNA was similar to that evoked by chronic E₂; i.e., endometria contained no OTR mRNA, and corresponding concentrations of OTR were too low to measure with accuracy. On this basis, one might conclude that the effect of chronic E₂ treatment of ewes on OTR gene transcription is indeed mediated by P₄. The ability of this steroid to suppress OTR gene transcription is consistent with the report of Stewart et al. [27], who found an absence of endometrial OTR mRNA in the ewe during the P₄-dominated luteal phase of the cycle. The mechanism by which P₄ acts to suppress transcription of the OTR gene is not known. Although we cannot exclude the possibility of a nongenomic action of P₄ [12], the lack of OTR mRNA in P₄-treated animals suggested that this steroid inhibits OTR gene transcription. However, there was no significant difference in the endometrial concentration of cytoplasmic PR between control and P₄-treated ewes. Cytoplasmic concentrations of PR were lower than those reported to be present during the midluteal phase of the cycle of the ewe [28], and nuclear concentrations of PR were below the detection limits of the assay. On the basis of the data of Wathes et al. [29], it is likely that the PR measured on Day 15 in P₄-treated ewes were localized predominantly in the caruncular stroma. On Day 14 of the cycle, OTR are found primarily in the uterine luminal epithelium [29, 30]. If one accepts this apparent difference in tissue distribution of OTR and PR, then it is logical to propose that P₄ acts on stromal cells to stimulate the production of some factor capable of suppressing OTR gene expression in the luminal epithelial cells. The fact that OTR apparently increase after prolonged treatment of ovariectomized ewes with P₄ [31] may be due to eventual accumulation of E₂ resulting from the peripheral conversion of the P₄ to this estrogen [32].

In view of the possible mode of action of P₄ described above, it is conceivable that the suppressive effects of chronic E₂ and P₄ on OTR gene expression likely involve independent mechanisms. For example, in intact ewes [7] and cows [33], endometrial concentrations of OTR are maximal at estrus as systemic concentrations of E₂ are increasing and then decline by Days 3–5 of the cycle. The reduction in uterine OTR cannot be attributed to inhibition by P₄ because luteal production of this steroid is minimal at these times. Further, a similar response to E₂ has been observed to occur in ovariectomized cows after pretreatment with P₄ to mimic the natural luteal phase of the cycle [34] and in ovariectomized ewes given no previous P₄ treatment [31]. These data suggest that in the intact ewe and cow, follicular secretion of E₂ at estrus causes down-regulation of uterine OTR, perhaps by the same mechanism as chronically administered E₂ in the ewe.

Flint and Sheldrick [1] were the first to demonstrate that an exogenous analogue of PGF₂ induced the secretion of luteal OT in ewes during the late stages of the estrous cycle. Because ovine large luteal cells are the source of OT and also contain ER [14], it was hypothesized that chronic treatment of ewes with E₂ may alter luteal production/secretion of this nonapeptide. However, results of the present study indicate that chronic treatment of ewes with E₂ did not affect PGF₂-induced secretion of OT on Day 15. Treatment with E₂ appeared to result in a sustained release of OT in response to PGF₂ relative to the value in controls, but this could simply be a reflection of the sampling frequency. These data suggest that although chronic E₂ treatment interferes with luteolysis by down-regulating OTR, the PGF₂-inducible component of the putative positive feedback system in these animals is fully functional.

In conclusion, it appears that chronic treatment of ewes with E₂ during the entire cycle, or treatment of ewes with P₄ only during the late luteal phase of the cycle, suppresses OTR gene transcription with a concomitant reduction in endometrial concentration of the receptor. Data from this study suggest that the suppressive effects of each steroid on endometrial OTR are the consequence of each hormone acting through a distinctly different mechanism. Further, at this time, there is no evidence that chronic E₂ treatment of ewes during the cycle has any impact on the ability of the CL to secrete OT.

	ACKNOWLEDGMENTS

 
The authors would like to thank Gordon Niswender, Colorado State University, Fort Collins, CO, for the P₄ antiserum; D. Schams, Technical University Munich, Freising-Weihenstephan, Germany, for the OT antiserum; Dr. Tadashi Kimura, Osaka University Medical School, Osaka, Japan, for the OTR cDNA; Ov Slayden, Oregon Regional Primate Center, Beaverton, OR, for his help with the PR analysis; Jennifer Bertrand, Iowa City, IA, for her advice in performing the OT RIA; Viola Manning for preparation of the 18S rRNA probe and her advice in preparing the OTR mRNA; David Thomas, Department of Statistics, for his assistance in the statistical analysis of these data; and Clint Abbe and Christine Colvin for their help with animal handling and laboratory techniques.

	FOOTNOTES

 
¹ Technical paper no. 11,220, Oregon Agricultural Experiment Station.

² Correspondence: F. Stormshak, Dept. of Animal Science, Oregon State University, Corvallis, OR 97331-6702. FAX: (541) 737–4174;stormshf{at}ccmail.orst.edu

Accepted: February 25, 1998.

Received: May 12, 1997.

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