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Regular Article |
from Bovine Endometrial Tissue1
a Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, 10-718 Olsztyn, Poland
b Department of Animal Sciences, University of Kentucky, Lexington, Kentucky 40546-0215
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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from bovine endometrial tissue explants was examined. Endometrial tissue from the late luteal phase were preincubated for 20 h in control medium. Explants were then treated for 6 h with control medium, oxytocin (10-7 M), progesterone (10-5 M), or both hormones. Oxytocin increased the medium concentration of 13,14-dihydro-15-keto-PGF2, whereas progesterone completely suppressed the stimulatory effect of oxytocin. In experiment 2, isolated endometrial epithelial cells were incubated with progesterone (10-5 M), oxytocin (10-7 M), and combinations of these hormones with or without actinomycin D (1 ng/ml). Only oxytocin stimulated secretion of PGF2, and this response was suppressed by progesterone. Oxytocin induced a rapid increase in intracellular concentrations of Ca2+ detected within 1 min of exposure of epithelial cells from the same cows. Progesterone pretreatment diminished this response. In experiment 3, direct effects of progesterone (2 nM–20 µM) on binding of 3H-oxytocin to the membrane preparation from epithelial cells were determined by saturation analysis. Oxytocin binding was suppressed by progesterone at every dosage tested. Progesterone is capable of suppressing the ability of oxytocin to induce endometrial secretion of PGF2. This effect appears to be mediated through a direct interference in the interaction of oxytocin with its own receptor.
female reproductive tract, mechanisms of hormone action, oxytocin, progesterone, uterus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(the endogenous luteolysin) from the bovine uterus [1–3]. Ovarian steroids also affect uterine PGF2 secretion by acting more slowly to influence uterine secretory responsiveness to oxytocin. Stimulatory effects of progesterone on oxytocin-induced PGF2 secretion are observed after 
7 days of progesterone exposure [4, 5]. These effects appear to be exerted through transcriptional activation of genes that code for hormone receptors [5] and possibly PG synthesizing enzymes [6, 7]. However, apparent inhibitory effects of progesterone on uterine PG secretion have also been observed. In cattle, an acute suppression of peripheral concentrations of progesterone, either by manual enucleation of the corpus luteum or by inducing premature luteolysis with cloprostenol, leads to a rapid increase in PGF2 secretion from the uterus [8]. Similar responses have been demonstrated in sheep [9] and are due in part to a rapid increase in uterine secretory responsiveness to oxytocin [10]. In recent studies [11], the PGF2 secretory responsiveness of bovine endometrial tissue to oxytocin in vitro was completely suppressed by simultaneous treatment with progesterone. The objectives of the experiments described here were to characterize the in vitro inhibitory effect of progesterone on the PGF2 secretory responsiveness of bovine endometrial tissue to oxytocin in more detail and to determine how this effect is exerted at the cellular level. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Uteri were obtained from nonpregnant cows slaughtered at a local abattoir and were transported to the laboratory in ice-cold PBS within 1 h of death. The stage of the estrous cycle was assessed by morphological observations of the reproductive tract [12, 13]. Uteri from cows determined to be in the late luteal stage of the cycle (16–18 days postestrus) were used in the present experiments. Endometrial tissue from the uterine horn ipsilateral to the ovary bearing the corpus luteum was used in all experiments.
Preparation and Culture of Endometrial Slices
After reaching the laboratory, uteri were washed 3 times in saline containing penicillin (100 IU/ml) and streptomycin (100 µg/ml). Endometrial tissue from the uterine horn ipsilateral to the corpus luteum was dissected from the underlying muscularis layer with a scalpel. The tissue was cut into small pieces (weighing approximately 30 mg) and washed in sterile saline. Individual endometrial slices were placed in culture vials and incubated in Dulbecco modified Eagle medium (DMEM; Sigma Chemical Co., St. Louis, MO) supplemented with 0.1% BSA, penicillin (100 IU/ml), and streptomycin (100 µg/ml) in a shaking water bath at 37°C in air with 5% CO2. After 20 h of incubation, medium was replaced with fresh medium containing treatments for an additional 6 h of incubation. Each treatment was applied to individual explant cultures in triplicate.
Isolation of Endometrial Epithelial Cells
Epithelial cells were separated by the procedure of Skarzynski et al. [14]. The uterine lumen was washed 3 times with 30–50 ml of Ca2+-free and Mg2+-free Hanks balanced salt solution (HBSS) containing 0.1% BSA (Sigma) and supplemented with 100 IU/ml penicillin and 100 µg/ml streptomycin. The lumen of the uterine horn was filled with HBSS containing 0.05% collagenase I (Sigma), 0.005% deoxyribonuclease I (Sigma), and 0.1% BSA. The open ends of the uterine horns were sealed using hemostat clamps. The sealed uterine horns were then incubated at 37°C for 45 min in a water bath. The enzyme mixture containing free cells was collected and replaced with fresh mixture, which was then incubated for an additional 30 min. The enzyme mixture was collected, pooled with the mixture from the previous incubation, and filtered through a metal screen (100-µm mesh) to separate cells from dissociated fragments of tissue. Free cells in the filtrate were washed 3 times with DMEM supplemented with antibiotics and 0.1% BSA. Cells were counted using a hemocytometer. Viability was estimated by exclusion of 0.04% trypan blue dye.
Culture of Endometrial Epithelial Cells
Cells were cultured in DMEM/Ham F-12 1:1 (v:v) medium (Sigma) supplemented with 10% calf serum and 20 mg/ml gentamicin. Cells were seeded at a density of 105 viable cells/ml in 48-well plates (Corning Glass Works, Corning, NY) and incubated at 37°C in a humidified atmosphere of 5% CO2 and 95% air. Cells were cultured for 48 h to permit attachment. Medium was then changed at 24-h intervals until cells were confluent (6–7 days). Confluent cells were then incubated in DMEM/Ham F-12 supplemented with 0.1% BSA, 5 ng/ml sodium selenite, 0.5 nM ascorbic acid, 5 µg transferrin, and 20 mg/ml gentamicin. Treatments were also applied at this time. Each treatment was applied to triplicate wells within replicates (cows).
Preliminary Experiment
This experiment was conducted to confirm that the stimulatory effect of oxytocin on PGF2 secretion that was detectable in 24-h cultures of endometrial tissue [11] could also be detected in cultures that lasted only 6 h. Endometrial slices were incubated in control medium or medium containing either of two concentrations of oxytocin (10-6 or 10-7 M). Secretion of PGF2 was determined by measuring the concentration of 13,14-dihydro-15-keto-PGF2 (PGFM) present in the culture medium [11]. PGF2 secretion by bovine endometrial slices was stimulated at both concentrations of oxytocin tested (P < 0.001; Fig. 1). Because the potency of oxytocin stimulation was similar at both concentrations of oxytocin (10-6 and 10-7 M), the lower concentration was chosen for further experiments.
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Experiment 1
The purpose of this experiment was to determine whether progesterone can diminish the stimulatory effect of oxytocin on PGF2 secretion from bovine endometrial tissue. Slices of endometrium in 4 replicates from 3 cows were treated with progesterone (10-5 M), oxytocin (10-7 M; Sigma), and combinations of these hormones. Treatments were applied during the final 6-h incubation period, and culture medium was collected for PGFM determination.
Experiment 2
The purpose of this experiment was to determine whether isolated endometrial epithelial cells respond directly to progesterone and oxytocin in a manner similar to that of the endometrial explants studied in experiment 1 and to establish whether the inhibitory effect of progesterone on oxytocin-stimulated PGF2 secretion is dependent upon de novo synthesis of RNA (transcription). Endometrial epithelial cells were incubated with 1) control medium, 2) oxytocin (10-7 M), 3) progesterone (10-5 M), 4) oxytocin and progesterone, 5) oxytocin and actinomycin D (1 ng/ml), or 6) oxytocin, progesterone, and actinomycin D. The concentration of actinomycin D that efficiently inhibited transcription was established in a previous experiment, where different concentrations of actinomycin D were challenged to inhibit genomic process of tumor necrosis factor -stimulated PGE2 secretion in bovine luteal cells [15]. Treatments were applied during the final 4-h incubation period, and culture medium was collected for measurement of PGF2 concentration by RIA. This experiment was replicated with epithelial cells from 3 cows.
Experiment 3
Mobilization of intracellular Ca2+ was evaluated using the cell-permeable form of the fluorescent Ca2+ indicator Fura-2 (Sigma) [16]. After the endometrial epithelial cells reached confluence, the medium was exchanged for DMEM/Ham F12 with 0.1% BSA, and cells were incubated for 24 h in a humidified atmosphere at 37°C. Cells were washed 3 times with M-199 medium, and 5 µM Fura-2 was added to the culture wells. The cells were incubated at 37°C for 40 min and then washed 4 times in M-199 medium. Cells were then incubated in DMEM/Ham F12 medium supplemented with 0.1% BSA for 30 min at 37°C to allow hydrolysis of cytoplasmic Fura-2. Cells were then washed 3 times in M-199. Changes in the intracellular concentrations of Ca2+ were monitored using an inverted microscope equipped with a fluorescent lamp and a Fura-2 filter. Every 10 sec, the intensity of fluorescence and the area occupied by fluorescencing cells were measured, from 10 sec before through 130 sec after treatment with oxytocin, progesterone, or oxytocin after 15 min of pretreatment with progesterone. Changes in intracellular Ca2+ concentrations following treatments were analyzed by computer software (Micro Image 4.0; Olympus Optical Co., Hamburg, Germany).
Experiment 4
The purpose of this experiment was to determine whether progesterone is able to reduce oxytocin binding to the oxytocin receptor. Oxytocin receptor concentrations were determined using the radioreceptor assay procedure. Progesterone (20, 2, 0.2, 0.02, and 0.002 µM) was added directly to the receptor incubation assay. Receptor concentrations were determined on membranes prepared from endometrial tissue from 4 cows.
Hormone Assays
Concentrations of PGF2 were determined directly in the medium [17] by enzyme immunoassay using peroxidase-labeled PGF2 as a tracer (1:40 000 final dilution; donated by Dr. K. Okuda, Okayama University, Okayama, Japan) and anti-PGF2 serum (1:5000; donated by Dr. W.W. Thatcher, University of Florida, Gainesville, FL). The PGF2 standard curve ranged from 0.16 to 20 ng/ml, and the ED50 of the assay was 0.45 ng/ml. The intra- and interassay coefficient of variations were 7.5% and 11.5%, respectively.
PGFM concentration was determined by the method described by Homanics and Silvia [18]. The sensitivity of the assay was 10 pg/ml, and the intra- and interassay coefficients of variation were 10.5% and 15.2%, respectively.
Radioreceptor Assay
Oxytocin receptors in endometrial tissue were quantified according to the procedure of Sheldrick et al. [19] with later modification by Mirando et al. [20]. Approximately 1 g of endometrial strips was incubated for 20 h in 20 ml DMEM/Ham F12 in a progesterone-free environment in a humidified atmosphere of 5% CO2 in air at 37°C to maximize oxytocin receptor number replenishment. After incubation, the medium was replaced with 10 ml of 1 nM EDTA and 0.9% NaCl (4°C) and rinsed with an additional 10 ml buffer. Buffer was replaced with 10 ml of 25 mM Tris-HCl and 250 mM sucrose (pH 7.4, 4°C), and tissue was mechanically homogenized (Ultra-Turax; IKA-Labortechnik, Staufen, Germany) at 24 000 rpm. Homogenates were filtered through 4 layers of cheesecloth into chilled ground-glass homogenizers and further homogenized with 10 strokes of the pestle. The homogenates were centrifuged for 10 min at 2000 x g at 4°C to precipitate large particulate debris and nuclei. The supernatants were gently placed in ultracentrifuge tubes and centrifuged for 90 min at 45 000 x g at 4°C to precipitate membranes. The membrane pellets were then rinsed twice with 5 ml of 25 mM Tris-HCl and 0.02% NaN3 (pH 7.4, 4°C) and resuspended in 4.5 ml of this buffer. The protein concentrations of the suspensions were determined using Bradford reagent (Sigma). Membrane preparations containing 50 µg protein were added to the tubes containing 0.05–8.0 pmol [3H]-oxytocin (New England Nuclear, Zaventem, Belgium) in 100 µl of 25 mM Tris-HCl, 20 mM MnCl2, 0.2% BSA, and 0.02% NaN3 (pH 7.4, 4°C) and incubated at 21°C. Nonspecific binding was determined by adding 8 nmol oxytocin. Samples were incubated at 21°C for 35 min, and then tubes were placed on ice. Two milliliters of 25 mM Tris-HCl, 10 mM MnCl2, 0.1% BSA, and 0.01% NaN3 (pH 7.4, 4°C) was added, and samples were filtered through 0.2-µm Durapore membrane filters (Millipore, Wien, Austria). Filters were rinsed with 2 ml of buffer, and receptor-bound [3H]-oxytocin retained on the filters was quantified by liquid scintillation counting (Beckman, Fullerton, CA).
Statistical Analysis
The data from experiments 1, 2, and 4 are presented as the mean (±SEM) values obtained in 3 or 4 cows. Mean values/bars of experimental groups were compared with each other by one-way ANOVA followed by a Bonferroni multiple comparison test, which compares all pairs of column data. Patterns of Ca2+ mobilization in experiment 3 were estimated by tests for repeated measures. Both tests were performed by computer using Prism 2 software (GraphPad Software, San Diego, CA).
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RESULTS |
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Concentrations of PGFM were higher in medium collected from explants treated with oxytocin than in medium collected from control explants (P < 0.01, Fig. 2). Progesterone did not affect PGFM concentrations when applied alone. However, when progesterone was added to the medium together with oxytocin, the stimulatory effect of oxytocin was suppressed (P < 0.01). The concentration of PGFM in that group was not different from that observed for control slices.
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Experiment 2
PGF2 production from the epithelial cells increased 3-fold compared with control in response to oxytocin (P < 0.01; Fig. 3). This stimulatory effect of oxytocin was reduced (P < 0.01) but not completely eliminated when progesterone was supplemented in the culture medium. Actinomycin D had no effect, either on the ability of oxytocin to stimulate PGF2 secretion or on the ability of progesterone to suppress the response to oxytocin.
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Experiment 3
Concentration of intracellular Ca2+ in endometrial epithelial cells was rapidly increased within 70 sec after oxytocin (10-7 M) treatment (Fig. 4A). However, Ca2+ mobilization was not observed (P > 0.05) in cells pretreated for 15 min with progesterone (Fig. 4A) as measured by area under the curve. Differences between these 2 treatment groups were not seen within the next 60 sec of measurements (Fig. 4B).
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Experiment 4
The specific [3H]-oxytocin binding capacity of endometrial membranes was 39.2 fmol/50 µg protein. Nonspecific binding averaged 33% of total binding. Specific binding of [3H]-oxytocin was suppressed by progesterone at all 5 concentrations examined (P < 0.01) with an efficiency comparable to a 1000-fold excess of cold oxytocin (Fig. 5).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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secretion by dispersed endometrial cells prepared from uteri judged to be from cows late in the luteal phase of the estrous cycle. These observations are in agreement with previous reports [11, 21]. Therefore, we believe that this is a reliable in vitro model with which to study the acute interactions of progesterone and oxytocin in regulating PGF2 secretion. In the present study, progesterone inhibited the stimulatory effect of oxytocin on endometrial PG secretion, confirming previous observations [11]. This effect of progesterone is exerted acutely, requiring no preincubation with progesterone prior to oxytocin exposure for the effect to be observed. The effect manifests itself in relatively short-term culture situations (6 h for studies using explants, 4 h for studies using endometrial cells). Therefore, these effects of progesterone probably are not mediated through genomic mechanisms, either activating or suppressing transcription of specific genes (e.g., the oxytocin receptor or PGH2 synthase 2). This effect of progesterone is extremely transient. Progesterone has no inhibitory effect on the response of cultured bovine uterine epithelial cells to oxytocin when administered for 72 h and then withdrawn for 6 h immediately prior to oxytocin exposure [21].
The mechanisms by which progesterone exerts its effect were partially elucidated in experiments 2, 3, and 4. In experiment 2, actinomycin D was used to show that the effect of progesterone was independent of new RNA synthesis, which implies that the inhibitory effect of progesterone does not depend on the transcriptional activation of specific genes. Blocking RNA synthesis, by itself, did not affect the ability of oxytocin to stimulate PGF2 secretion, implying that there is an adequate supply of oxytocin receptor mRNA to maintain oxytocin receptor numbers throughout the short-term culture period examined.
In experiment 3, oxytocin stimulated a rapid increase in intracellular Ca2+ concentrations in cultured bovine endometrial cells. This is the first demonstration of this effect in bovine uterine tissue. Burns et al. [22] showed that Ca2+ plays a very important role in mediating the stimulatory effect of oxytocin on PGF2 secretion by bovine endometrial tissue. However, preincubation of endometrial cells with progesterone for as little as 15 min inhibited oxytocin-stimulated intracellular Ca2+ mobilization. Thus, the inhibitory effect of progesterone may be mediated through its ability to disrupt this critical intracellular signaling pathway.
The concentration of progesterone used in experiments 1–3 (10-5 M) is high. However, in the next experiment we used a wide range of progesterone concentrations. In experiment 4, the ability of progesterone to inhibit binding of oxytocin to its receptor was clearly demonstrated. This effect was observed at concentrations of progesterone that fall within the normal physiological range, as low as 2 nM (approximately 0.6 ng/ml). This effect was exerted directly in membrane preparations, again implying that the effect does not require any genomic action of progesterone. This direct effect, at the level of the oxytocin receptor, can account for the reduction in the ability of oxytocin to stimulate both Ca2+ mobilization and PG secretion from bovine endometrial tissue. These results are consistent with those obtained by Grazzini et al. [23], who showed that oxytocin binding and oxytocin-stimulated calcium mobilization was suppressed by progesterone in a Chinese hamster ovarian cell line induced to express oxytocin receptors by transient mRNA transfection. Grazzini et al. also observed that the inhibitory effect of progesterone was maintained even when progesterone was conjugated to BSA. Precisely how oxytocin alters the oxytocin-receptor interaction is not clear. Picard [24] suggested that progesterone may bind the oxytocin receptor at an allosteric effector site and may induce a conformational change that prevents oxytocin from binding to its receptor. Alternatively, progesterone may bind to its own membrane receptor, as it appears to have done in Xenopus oocytes [25]. In this system, progesterone inhibits adenylate cyclase activity to induce resumption of meiosis. The activated progesterone-receptor complex may then interfere in oxytocin-receptor interactions.
The physiological implications of this direct inhibitory effect of progesterone on uterine secretory responsiveness to oxytocin are intriguing. Because uterine secretion of PGF2 is believed to initiate luteolysis, the initial release of PGF2 occurs in the face of high circulating concentrations of progesterone. Therefore, at least the first luteolytic pulse of PGF2 in cattle may not be induced by oxytocin as suggested [26]. Once luteolysis is initiated and progesterone concentrations begin to decline, oxytocin may play a role in the stimulation of later pulses of PGF2 [27]. Perhaps this is why the initial pulse(s) of PGF2, which temporally precedes luteolysis, is typically lower in magnitude than pulses that occur after progesterone concentrations have fallen to basal levels [28].
Progesterone suppresses the ability of oxytocin to induce endometrial secretion of PGF2. This effect appears to be mediated through a direct interference in the interaction of oxytocin with its own receptor; however, the detailed nature of this process in unknown.
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ACKNOWLEDGMENTS |
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and to Dr. W.W. Thatcher for donating the anti-PGF2 serum. |
FOOTNOTES |
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1 This research was financed by grant KBN 5P06K 04817 and supported by the Organization for Economic Cooperation and Development, Kentucky Agricultural Experiment Station. This research is published with the approval of the Kentucky Agricultural Experiment Station (publication 01-07-80). 
2 Correspondence. FAX: 48 89 524 0347; janko{at}pan.olsztyn.pl
Accepted: February 2, 2002.
Received: August 10, 2001.
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in heifers. Biol Reprod 1985; 33:1113-1119[Abstract] induced by oxytocin and endogenous concentrations of estradiol and progesterone at three stages of the bovine estrous cycle. J Anim Sci 1989; 67:2347-2353 in heifers. J Reprod Fertil 1988; 82:429-436 production in cows by progesterone and oestradiol. J Reprod Fertil 1995; 103:69-73 during the estrous cycle. Acta Vet Scand Suppl 1981; 77::143-158[Medline] in ewes following luteal regression induced with a prostaglandin analogue, estrumate (cloprostenol; ICI 80996). Prostaglandins 1976; 11:537-543[CrossRef][Medline] in response to oxytocin in ewes. Reprod Fertil Dev 1997; 9:255-262[CrossRef][Medline] secretion by the bovine endometrium in vitro. Theriogenology 1999; 52::385-397[CrossRef][Medline] a trigger for the initiation of endometrial prostaglandin F2 release at luteolysis in cattle?. Biol Reprod 2000; 62:1109-1115 by cultured bovine endometrial cells in response to tumor necrosis factor : cell type specificity and intracellular mechanisms. Biol Reprod 2000; 62:1116-1120 in response to oxytocin in ovariectomized ewes. Biol Reprod 1988; 38:723-728[Abstract] secretory response in uterus from ovariectomized ewes and activation of the response in vitro. J Endocrinol 1995; 145:299-305 secretion in vitro, and plasma concentrations of ovarian steroids before and during corpus luteum regression in cyclic heifers. Biol Reprod 1993; 48:874-882[Abstract] and E2 and response to oxytocin in cultured epithelial and stromal cells of the bovine endometrium. Biol Reprod 1996; 54:371-379[Abstract] synthesis in bovine endometrium: role of calcium. Domest Anim Endocrinol 1998; 15:477-487[CrossRef][Medline] in initiated luteolysis in cattle. Res Vet Sci 1999; 66:1-5[CrossRef][Medline] in the peripheral circulation during normal luteolysis in heifers. Prostaglandins 1976; 11:871-878[CrossRef][Medline]This article has been cited by other articles:
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) or oxytocin after pretreatment for 15 min with progesterone (10-5 M; 
) within the first 70 sec (A) and within the next 60 sec (B). Data are from a set of cells from 1 representative uterus. Similar profiles were obtained in 3 other experiments. Arrows indicate the time of treatment addition. Results for the progesterone pretreatment group are significantly different from those of the nonpretreated group (P < 0.01)