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Articles |
Is Dependent on Progesterone, Oxytocin, and Prostaglandins1
a Laboratory of Reproductive Endocrinology, Faculty of Agriculture, Okayama University, Okayama 700–8530, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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that is released from the uterus is essential for spontaneous luteolysis in cattle. Although PGF2 and its analogues are extensively used to synchronize the estrous cycle by inducing luteolysis, corpora lutea (CL) at the early stage of the estrous cycle are resistant to the luteolytic effect of PGF2. We examined the sensitivity of bovine CL to PGF2 treatment in vitro and determined whether the changes in the response of CL to PGF2 are dependent on progesterone (P4), oxytocin (OT), and PGs produced locally. Bovine luteal cells from early (Days 4–5 of the estrous cycle) and mid-cycle CL (Days 8–12 of the estrous cycle) were preexposed for 12 h to a P4 antagonist (onapristone: OP; 10-4 M), an OT antagonist (atosiban: AT; 10-6 M), or indomethacin (INDO; 10-4 M) before stimulation with PGF2. Although OP reduced P4 secretion (p < 0.001) only in early CL, it reduced OT secretion in the cells of both phases examined (p < 0.001). OP also reduced PGF2 and PGE2 secretion (p < 0.01) from early CL. However, it stimulated PGF2 secretion in mid-cycle luteal cells (p < 0.001). AT reduced P4 secretion in early and mid-cycle CL (p < 0.05). Moreover, PGF2 secretion was inhibited (p < 0.05) by AT in early CL. The OT secretion and the intracellular level of free Ca2+ ([Ca2+]i) were measured as indicators of CL sensitivity to PGF2. PGF2 had no influence on OT secretion, although [Ca2+]i increased (p < 0.05) in the early CL. However, the effect of PGF2 was augmented (p < 0.01) in cells after pretreatment with OP, AT, and INDO in comparison with the controls. In mid-cycle luteal cells, PGF2 induced 2-fold increases in OT secretion and [Ca2+]i. However, in contrast to results in early CL, these increases were magnified only by preexposure of the cells to AT (p < 0.05). These results indicate that luteal P4, OT, and PGs are components of an autocrine/paracrine positive feedback cascade in bovine early to mid-cycle CL and may be responsible for the resistance of the early bovine CL to the exogenous PGF2 action.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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released from the uterus has been shown to cause regression of the corpora lutea (CL) of many species, the mechanism of its action in inducing luteolysis remains unclear. PGF2 is released from the uterus at the end of the luteal phase as a series of pulses and reaches the ovary via countercurrent transfer in the utero-ovarian broad ligament [1]. Moreover, PGF2 and its analogues have been used extensively to synchronize the bovine estrous cycle [2]. However, newly formed CL of many mammalian species, including cattle, are resistant to the single treatment with exogenous PGF2 [3, 4]. The sensitivity of bovine CL to PGF2 seems to increase progressively toward the end of the luteal phase [5]. The ovine [6] and bovine CL [5] exhibit a differential sensitivity to PGF2 in terms of an increase of oxytocin (OT) secretion, indicating the presence of intracellular mechanisms that regulate different reactions of CL to PGF2.
If PGF2-induced luteolysis is a receptor-mediated phenomenon, the changes in the sensitivity of CL to PGF2 seem to depend on the density and affinity of the PGF2 receptor in CL toward the end of the luteal phase [7]. However, Sakamoto et al. [8, 9] showed that the expression of PGF2 receptor mRNA and the number and affinity of PGF2 binding sites were only slightly increased from the early to the late luteal phase of the bovine estrous cycle. Moreover, high-affinity PGF2 binding sites have been detected on the luteal membrane from early bovine CL [9]. This is in agreement with the results of Wiltbank et al. [10], which showed that early and active CL were not different with respect to PGF2 receptor concentration and affinity. Although one could assume that the unresponsiveness of early CL to PGF2 is not due to a lack of high-affinity PGF2 receptors, the mechanisms responsible for these facts are not yet clear.
We hypothesized that intraluteal factors may be responsible for the differing sensitivity of bovine luteal cells with respect to PGF2 action during the estrous cycle in cattle. It is well known that the bovine CL is a site of progesterone (P4), PG [11], and OT production [12]. Moreover, in addition to the presence of PGF2 receptors [13], high-affinity binding sites for OT [14] and P4 [15] have also been demonstrated in bovine CL. All these luteal factors may act on CL either independently or in concert to modify the actions of one another. Therefore, the present study was undertaken to determine the differing sensitivities of cultured bovine luteal cells isolated from early and mid-CL to PGF2, and to determine whether the changes of the CL response to PGF2 are attributable to P4, OT, and luteal PGs. Since PGF2 rapidly stimulates OT secretion [5] as well as increasing intracellular free Ca2+ ([Ca2+]i) [16] in a dose- and threshold-dependent fashion, we measured both OT and [Ca2+]i levels as indicators of CL sensitivity to PGF2.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ovaries with a CL were collected from Holstein cows at a local abattoir within 10–20 min after exsanguination and were submerged in ice-cold physiological saline before being transported to the laboratory. The stage of the estrous cycle was defined by macroscopic observation of the ovaries and the uterus [17, 18]. Dissociation of the luteal tissue and culture of luteal cells were performed as previously described [14]. Briefly, CL were perfused for 15 min with EGTA-buffer (0.1 mM EGTA; Sigma Chemical Co., St. Louis, MO; #E-4378), 10 mM Hepes (Sigma; #H-9136), 140 mM NaCl, 7.1 mM KCl (pH 7.4) to remove vascular blood and to loosen the connection between the vascular endothelial cells. Then, CL were perfused for 15 min with wash buffer (10 mM Hepes, 140 mM NaCl, 7.1 mM KCl, 5.0 mM CaCl2; pH 7.4). These perfusion buffers were bubbled with 5% CO2:95% O2 during perfusion. The dissociation of the cells was achieved by perfusing for 30 min with wash buffer containing 0.05% (w:v) collagenase (Sigma; #C-0130) and 0.1% (w:v) BSA (Boehringer Mannheim GmbH, Mannheim, Germany; #735078). The cells were dispersed from the CL matrix with steel combs to remove connecting tissues. Finally, the dissociated luteal cells were pooled and stirred for 30 min in Dulbecco's Modified Eagle's medium (DMEM, Sigma; #D-1152) containing 0.05% collagenase, 0.005% DNase I (Sigma; #D-5025), and 0.1% BSA in a water bath at 37°C. After stirring, cells were filtered through metal wire meshes (200 µm, 150 µm, and 80 µm) to remove undissociated tissue fragments. The filtrate was washed three times by centrifugation for 10 min at 50 x g with the DMEM, supplemented with 60 µg/ml penicillin, 100 µg/ml streptomycin, and 0.1% BSA. The cells were counted with a hemocytometer. Cell viability was higher than 85% as assessed by trypan blue exclusion. The cell suspension contained about 10% endothelial cells or fibrocytes and no erythrocytes. The cell suspension obtained from early CL contained about 10% large luteal cells and 80% small luteal cells. In mid-cycle CL, the number of large luteal cells increased approximately two times; however, the number of small luteal cells decreased by about 10%.
Cell Culture and Experiments
Although there is a study on the regulation of OT secretion in cultured bovine luteal cells [19], a number of workers have suggested that isolated bovine and ovine luteal cells in long-term monolayer culture lose an ability to produce OT (reviewed in [20]). Therefore, first we established a method to study the OT production in cultured bovine luteal cells. Cells prepared as described above were adjusted to 2.5 x 105 viable cells/ml of cultured medium: DMEM and F-12 Ham's medium (DMEM/Ham's F-12; 1:1 [v:v]; Sigma; #D-8900) supplemented with 0.1% BSA, 5 ng/ml sodium selenite, 0.5 mM ascorbic acid, 5 µg/ml transferrin, and 20 µg/ml gentamicin. The cells were incubated in glass culture tubes (12 x 75 mm) in a shaking water bath at 37.5°C. The culture medium was continuously gassed with 5% CO2 in air. The cells were simultaneously cultured and stimulated with various stimulators in the experiments to be described.
Experiment 1
We examined the possible effects of P4, OT, and PGs produced locally in CL on P4, OT, PGE2, and PGF2 secretion. Cells from early-cycle CL (Days 4–5 of the estrous cycle; 4 separate experiments, 2–3 CL in each experiment) and mid-cycle CL (Days 8–12 of the estrous cycle; 4 separate experiments, 2–3 CL in each experiment) were incubated in glass tubes in 2 ml culture medium for a total of 16 h with solvent only (control: 20 µl of 10% dimethyl sulfoxide; DMSO) or solvent with either a highly specific P4 antagonist (onapristone: OP; Schering AG, Berlin, Germany; #ZK98.299; 10-4 M), a highly specific OT antagonist (atosiban: AT; 1-deamino-2-D-Tyr(0ET)-4-Thr-8-Orn-vasotocin/oxytocin; Ferring AB, Malmö, Sweden; #CAP518; 10-6 M), or a selective cyclooxygenase inhibitor (indomethacin: INDO; Sigma; #I-7378; 10-4 M). After 12 h of incubation, the medium was replaced, after centrifugation for 5 min at 50 x g, by fresh medium with 0.1% BSA containing fresh OP, AT, or INDO. Samples were collected from the last 4 h of culture and stored at -30°C until the P4, OT, PGF2, and PGE2 could be determined. Doses of reagents were defined by preliminary experiments (data not shown) based on previous reports of others and ourselves [11, 21–23]. The concentrations of OP (a highly specific P4 antagonist) [24] and AT (a highly specific OT antagonist) [21] used in the present study are approximately 100 times higher than the affinities of P4 [15] and OT receptors [14] in bovine CL, and are approximately 10 times higher than production of P4 and OT in cells stimulated with LH (100 ng/ml) and PGF2 (10-6 M) for 16 h (data not shown). The viability of control and OP-, AT-, and INDO-treated cells after 16 h of incubation was similar (96 ± 2%, 93 ± 3%, 95 ± 1%, and 95 ± 2%, respectively) as assessed by trypan blue exclusion.
Experiment 2
We examined the possible effects of OT, PGs, and P4 produced locally in CL on PGF2-stimulated OT secretion. Cells produced in the same way as for experiment 1 were used for this experiment. Cells from early (4 separate experiments, 2–3 CL in each experiment) and mid-cycle CL (4 separate experiments, 2–3 CL in each experiment) were preincubated in glass tubes in 2 ml culture medium for 12 h with solvent only (control: 20 µl of 10% DMSO) or solvent with either a highly specific P4 antagonist (OP; 10-4 M), a highly specific OT antagonist (AT; 10-6 M), or a selective cyclooxygenase inhibitor (INDO; 10-4 M). After 12 h of incubation, the medium was replaced by fresh medium with 0.1% BSA containing PGF2 at a dose of 10-6 M and fresh OP, AT, or INDO. After an additional 4 h of incubation, culture media were collected and stored at -30°C until the P4, OT, PGF2, and PGE2 could be determined.
Experiment 3
We evaluated the influence of P4, luteal OT, and PGs on PGF2-mobilized free cytosolic [Ca2+]i. The luteal cells were prepared as described above from early-cycle CL (4 separate experiments, 2–3 CL in each experiment) and mid-cycle CL (3 separate experiments, 2–3 CL in each experiment) and were adjusted to 5.0 x 105 viable cells/ml before incubation for 12 h in glass tubes in 3 ml culture medium containing solvent only (control: 30 µl of 10% DMSO) or solvent with either OP (10-4 M), AT (10-6 M), or INDO (10-4 M). After 12 h of incubation, the medium was replaced after centrifugation as described above with Hanks' Balanced Salt Solution (Sigma #H-2387; pH 7.4) supplemented with 0.14 g CaCl2/L and 0.1% BSA, and [Ca2+]i was determined.
Hormone Determination
Measurement of P4 in the culture media was performed using a direct enzyme immunoassay (EIA) as described previously [25]. Antiserum to P4 (OK-1) was used at a final dilution of 1:600 000. The standard curve ranged from 0.39 to 100 ng/ml, and the effective dose for 50% inhibition (ID50) of the assay was 4.5 ng/ml. The intra- and interassay coefficients of variation were 5.4% and 8.6%, respectively.
The concentration of PGF2 was also determined directly in the media by EIA as described previously [26]. The PGF2 standard curve ranged from 15.625 to 4000 pg/ml, and the ID50 of the assay was 250 pg/ml. The intra- and interassay coefficients of variation were 7.4% and 11.6%, respectively.
PGE2 concentration was determined with EIA as described previously [27]. The PGE2 standard curve ranged from 0.110 to 28.2 ng/ml, and the ID50 of the assay was 0.97 ng/ml. The intra- and interassay coefficients of variation were 3.7% and 7.4%.
The EIA for OT was based on the second-antibody method using the biotin-streptavidin-peroxidase technique as described by Miyamoto et al. [28]. Anti-rabbit OT antiserum (R-1) showed less than 0.01% cross-reactivity with arginine-vasopressin, lysine-vasopressin, angiotensin, vasotocin, and somatostatin. In brief, 50-µl aliquots of standards and samples of culture medium obtained during experimentation were incubated at 4°C for 18–24 h with 100 µl OT antiserum (final concentration 1:150 000) in duplicates in 96-well ELISA plates (Corning Inc., Corning, NY) coated with goat anti-rabbit secondary antibody (anti-rabbit IgG; Seikagaku Co., Tokyo, Japan). After reagents were discarded, the biotinylated OT was added to 100 µl EIA buffer. The plates were next incubated for 2 h and decanted, and 20 ng of streptavidin-peroxidase in 100 µl EIA buffer was added. After 15 min of incubation at 4°C, all wells were washed four times with 300 µl 0.05% Tween 80; and after addition of 150 µl substrate buffer with 0.05% 3,3',5,5'-tetramethylbenzidine (Wako Chemicals Co., Osaka, Japan) to each well, the plates were further incubated at 36°C for 40 min in the dark. The reaction was stopped by addition of 50 µl of 2 M H2SO4 to each well. The absorbance was measured at 450 nm with a plate reader (Bio-Rad, Hercules, CA). The standard curve ranged from 3.91 to 1000 pg/ml, and the ID50 of the assay was 41.3 pg/ml. The intra- and interassay coefficients of variation were 5.7% and 9.4%, respectively.
Measurement of [Ca2+]i Concentration
Intracellular Ca2+ concentrations were determined by use of the fluorescent Ca2+ indicator Fura-2 [29]. After 12 h of incubation with or without OP, AT, and INDO, cells were centrifuged (5 min at 50 x g) and washed and resuspended in Hanks' solution. Fura-2 AM (Dojindo, Kumamoto, Japan; #348–05831), the lipophilic acetoxymethylester form of Fura-2, was dissolved in DMSO to form a 1 mM stock solution; and 10 µl was added to 2-ml cell suspensions (final concentration 5 µM) to preload the cells with dye. The cells were incubated at 37°C for 40 min and then washed three times in Hanks' solution. After washing, the cells were postincubated for 30 min in Hanks' solution at room temperature to ensure full hydrolysis of the Fura-2 ester. Spectrofluorometric measurements were conducted in 1.5-ml samples continuously stirred in a quartz-glass cuvette and thermostatically maintained at 37°C. Fluorescence was monitored using a Shimadzu spectrofluorometer RF-5000 (Shimadzu, Kyoto, Japan). PGF2 (10-6 M) dissolved in DMSO, and DMSO only as a control, were added (15 µl) into the cuvette through a port in the sample compartment connected to a tuberculin syringe. Excitation and emission wavelengths were 340 nm and 490 nm, respectively, with slit widths of 5 nm for both wavelengths. Intracellular [Ca2+]i concentrations were calculated from the equation:
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The KD for Fura-2 at 37°C was taken to be 224 nM [29]. Maximum and minimum fluorescence (Fmax and Fmin) were measured by rapidly saturating Fura-2 with Ca2+ by permeabilizing the cells with 0.2% Triton X-100 (Fmax) and by adding 5 mM EGTA in Tris-HCl buffer, pH 8.5, to determine the basal fluorescence (Fmin) when virtually no Ca2+ was bound to Fura-2.
Statistical Analysis
The data are shown as the means ± SEM of 3–4 separate experiments each performed in triplicate. Because OP, AT, and INDO influence the basal rate of OT secretion, OT was expressed as a percentage of an internal standard. The baseline was removed by using the computer program PRISM (GraphPad Software, San Diego, CA). The total PGF2-induced increase in [Ca2+]i in the cells pretreated or not pretreated with OP, AT, and INDO was measured by calculating the area under the curve (PRISM). The baseline for [Ca2+]i was defined based on data from the resting period before PGF2 or DMSO treatment (see Fig. 4). The statistical significance of differences between controls and treated groups was assessed by one-way ANOVA followed by Bonferroni's Multiple Comparison test (PRISM).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, and PGE2 by Bovine Luteal Cells Incubated with a P4 Antagonist (OP), an OT Antagonist (AT), and INDO
The concentrations of P4, OT, PGF2, and PGE2 released from bovine luteal cells, cultured in glass tubes in the presence or absence of OP, AT, and INDO, are shown in Figure 1. Bovine LH (USDA-bLH-B-6) stimulated P4 secretion (p < 0.001) from cultured luteal cells on Days 4–5 and 8–12 of the estrous cycle, indicating that the cells cultured with the present experimental design were reactive (Fig. 1a). Although OP reduced P4 secretion (to 62.7% of the baseline; p < 0.001) only in early CL (Fig. 1a), it reduced OT secretion in the luteal cells of both early and mid-cycle CL (to 52.4% and 55.5% of the baseline, respectively; p < 0.001, Fig. 1b). As for secretion of PGs, the inhibition of P4 action by OP on luteal cells evoked reactions that depended on the day of the estrous cycle (Fig. 1, c and d). Furthermore, although OP reduced PGF2 and PGE2 secretion (to 50.5% and 65.6% of the baseline, respectively; p < 0.01) from early CL, it stimulated PGF2 secretion in mid-cycle luteal cells (to 207% of the baseline; p < 0.001). AT reduced P4 secretion (Fig. 1a) in early and mid-cycle CL (to 76.8% and 81.1% of the baseline, respectively; p < 0.05). Moreover, in early CL (Fig. 1c) in the presence of AT, PGF2 secretion was significantly lower compared with that in the control group (p < 0.05). In contrast to AT, INDO inhibited the secretion of both PGs in both the early and mid-cycle luteal cells (p < 0.001; Fig. 1, c and d).
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P4 and OT Secretion in Response to PGF2 Treatment in Bovine Luteal Cells Preincubated with a P4 Antagonist (OP), an OT Antagonist (AT), or INDO
Although PGF2 did not stimulate P4 secretion (p > 0.05) from cultured cells in either phase (Fig. 2), pretreatment of the early luteal cells with OP, AT, or INDO augmented the effect of PGF2 on the stimulation ratio for P4 to 186%, 168%, and 176% (p < 0.05), respectively, compared to value in the control group. In contrast with results for early CL, PGF2 stimulated P4 secretion (182%; p < 0.05) when the cells of mid-CL were preincubated only with OT antagonist. The effects of PGF2 on OT secretion in the presence or absence of either AT, OP, or INDO in early and mid-cycle CL are shown in Figure 3. PGF2 significantly increased OT secretion only in mid-luteal CL (to 195% of the baseline; p < 0.05, Fig. 3b). When the cells were pretreated with AT, PGF2 augmented OT secretion to 239% of the baseline in early CL (p < 0.001) and to 346% of the baseline in mid-cycle CL (p < 0.001). Moreover, the effect of PGF2 was raised in the cells of early-cycle CL pretreated with OP (to 262% of the baseline; p < 0.001) and INDO (to 220% of the baseline; p < 0.01).
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Cytosolic Free Ca2+ in Bovine Luteal Cells Preincubated with a P4 Antagonist (OP), an OT Antagonist (AT), or INDO in Response to PGF2 Treatment
The mean resting level of [Ca2+]i in bovine luteal cells before PGF2 addition was approximately 36.2 nM in 4 separate cell preparations from early CL and 3 preparations from mid-cycle CL. While preincubation of the cells with INDO elevated the resting level of [Ca2+]i (43.3 nM; p < 0.05), OP inhibited it (31.7 nM; p < 0.05) in comparison to values in both DMSO-pretreated groups (Fig. 4). DMSO did not stimulate the [Ca2+]i level (p > 0.05) in cultured cells in either of the estrous phases examined. The increases of [Ca2+]i observed in response to PGF2 from one representative experiment with early CL are shown in Figure 4. The treatment with PGF2 (10-6 M) resulted in two phases in the [Ca2+]i response, i.e., a rapid and transient rise immediately after the addition of PGF2 (initial phase), followed by a sustained secondary increase (influx of extracellular Ca2+). The duration of the initial phase was 10–50 sec (the period from the rise to the decay of the peak). In the early-cycle luteal cells preincubated with DMSO, PGF2 increased [Ca2+]i only to 146% of the baseline (Table 1). However, pretreatment of the cells with OP (Fig. 4a), AT (Fig. 4b), or INDO (Fig. 4c) augmented the effect of PGF2 on the stimulation of [Ca2+]i, resulting in [Ca2+]i levels of 284%, 228%, and 242% of the baseline (p < 0.05), respectively (Table 1). In mid-cycle luteal cells preincubated with DMSO, only PGF2 induced a 2-fold increase in [Ca2+]i (Table 1). However, in contrast to the PGF2-induced increase in [Ca2+]i in early CL, the PGF2-induced increase in [Ca2+]i in mid-cycle CL was magnified only by preincubating the cells with OT antagonist (to 276% of the baseline; p < 0.05).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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treatment during the estrous cycle are dependent on locally produced hormones. We measured P4 and OT secretion as well as [Ca2+]i as the indicators of responsiveness of CL to PGF2 actions. PGF2 stimulated both P4 (Fig. 2) and OT (Fig. 3) production in cocultured dispersed bovine luteal cells. These results confirmed previous data showing that PGF2 may have a luteotropic effect in small luteal cells but did not prove to be luteolytic in large luteal cells in vitro [19, 30]. Moreover, some differences in the cell response to PGF2 actions have been suggested between large and small luteal cells [31, 32]. Although PGF2 also increased [Ca2+]i in cocultured dispersed luteal cells (Fig. 4; Table 1), the stimulation ratio and resting level of [Ca2+]i in our study are considerably lower than those reported previously [31, 32]. These discordant results might be attributable to differing methodology, e.g., use of 12- and 16-h incubation periods, use of different cell numbers and proportions, and use of different doses of PGF2. Moreover, preincubation of the cells with OP slightly inhibited the resting level of [Ca2+]i, whereas INDO elicited Ca2+-mobilizing effects (Fig. 4) as reported by Alila et al. [31].
In the present study, although the rates of OT secretion in early CL (Fig. 3a) were not influenced by PGF2, pretreatment with OP, AT, or INDO resulted in increases in OT secretion. In contrast to the results from early CL, the sensitivity of mid-cycle CL to PGF2 significantly increased only after blockade of the OT receptors (Fig. 3b). Although there have been a few studies on the mechanisms responsible for the insensitivity of early CL to the action of PGF2 in cattle [10, 33], the insensitivity seems not to be due to a lack of high-affinity PGF2 receptors on either small or large bovine luteal cells [9, 13]. The response of CL to PGF2 depends on the activation of membrane receptors and an intracellular signaling system in the luteal cells [8, 16]. Therefore, we assumed that the lack of response to PGF2 in early CL could be a consequence of receptor desensitization as a general adaptive tendency of biological responses to wane over time. Desensitization of receptors is characterized by loss of receptor responsiveness to a stimulus of constant intensity, including the regulation of receptor number and affinity but also an inability of the receptors to fully activate their second messenger systems [34, 35]. Data from experiment 3 strongly support this hypothesis (Table 1). Interestingly, although PGF2 only slightly increased [Ca2+]i in early luteal cells (to 146% of the baseline) (Fig. 4; Table 1), pretreatment of the cells with OP, AT, or INDO amplified the effect of PGF2 on the stimulation rates of [Ca2+]i to a level greater than 200% compared with the control value, as is shown to occur in mid-cycle luteal cells (Table 1). Therefore, all of the results strongly support the hypothesis that the different effects of PGF2 on bovine CL during the estrous cycle depend on locally produced P4, OT, and PGs.
Hormone-induced desensitization of G protein-coupled receptor has been divided into two general categories, referred to as agonist-specific (or homologous) desensitization and agonist-nonspecific (or heterologous) desensitization [34, 35]. In the present study, INDO strongly inhibited secretion of PGF2 in the luteal cells of both examined periods (Fig. 1, c and d). Moreover, OP and AT also inhibited PGF2 secretion in the early CL. Furthermore, the inhibition of PGF2 production in early CL raised the effect of exogenous PGF2 on both OT secretion (Fig. 3a) and [Ca2+]i (Table 1). PGs produced locally in CL have been suggested as a reason for the failure of PGF2 to stimulate additional secretion of OT from ovine CL [36, 37]. In live ewes, low-level infusion of PGF2 into the ovarian artery desensitized the CL in terms of OT release, and a rest period of 6 h was required to restore the normal OT response to PGF2 [38]. On the other hand, twice-daily i.m. injections of exogenous PGF2 on Days 3 and 4 of the estrous cycle induced precocious luteolysis in conscious cattle [3]. This suggests that PGF2 treatment might up-regulate PGF2 receptors in early CL, not down-regulate them. This supposition has been supported by a recent report [39] that the expression of PGF2 receptor in cows increased by 24 h of treatment with PGF2, remained elevated at 48 h, and declined by 72 h. However, it has been also reported that treatment of cows with PGF2 in one luteolytic bolus on Day 4 of the estrous cycle decreased mRNA for PGF2 receptor by 4 h [33]. Moreover, in long-term (24 h)-cultured bovine luteal cells, PGF2 induced a dose-dependent cleavage of its own receptor [13]. Therefore, our data along with previous findings suggest that homologous desensitization, including down-regulation [13, 33, 39] of PGF2 receptors in bovine CL, might be due to long-lasting stimulation by luteal PGF2, particularly in early CL [11, 40].
However, the possibility of heterologous regulation by other intraluteal factors (P4, OT, PGI2, and/or PGE2) cannot be excluded. The present study demonstrated that OT as well as P4 plays some role as an autocrine/paracrine regulator in bovine CL during the early to mid-luteal phase. The OT antagonist inhibited P4 secretion in both examined periods (Fig. 1b). These results are in agreement with those of Sakumoto et al. [41], who demonstrated a significant effect of OT on P4 secretion by bovine luteal tissues. Moreover, the inhibition of the autocrine/paracrine OT action on luteal cells augmented the CL sensitivity to PGF2 (Figs. 2–4; Table 1). Therefore, it can be assumed that OT, through its luteotropic actions on early to mid-cycle CL, may indirectly (via PGF2) or directly (via heterologous desensitization) affect the functionality of PGF2 receptors and/or second messenger formation. In support of this hypothesis, OT receptors belong to the same G protein-coupled family as PGF2 receptors [42, 43], and common autoregulatory mechanisms involving the same secondary messenger cascade might cross-react with one another.
In the present study, we clearly demonstrated that P4 acts on the functionality of bovine early CL in an autocrine/paracrine fashion. In order to remove the influence of P4, which is produced during culture, OP was added to the cultured bovine cells. In the early luteal cells, secretions of P4 and of OT and also the production of both PGs were greatly reduced by OP (Fig. 1). Moreover, OP inhibited OT secretion in mid-cycle luteal cells, although it stimulated PGF2 secretion. These findings suggest that P4 appears to act in the early CL by stimulating P4, OT, and PG secretion but in the mid-cycle CL by inhibiting PGF2 secretion. Similarly, stage-of-cycle-dependent effects of P4 on PGF2 production have been suggested in the endometrium (reviewed in [1]). Thus, present data support the views [15, 44] that P4 may directly regulate the production of P4 [22], OT [23], and PGs [45] by the bovine ovary in a cycle-dependent fashion. Moreover, the present study also sheds light on the possible effects of P4 on PGF2 action in bovine CL. In the presence of OP, the action of PGF2 on luteal cells of the early stage was augmented (Figs. 2a, 3a, and 4a; Table 1). Therefore, one might assume that both OT and P4 are components of an autocrine/paracrine positive feedback cascade in bovine early to mid-cycle CL and that they play roles in regulating the functionality of PGF2 receptors and the intracellular calcium/protein kinase C cascade.
PGF2 and OT may play various roles in the regulation of luteal function at different phases of the estrous cycle. Stimulatory or inhibitory effects may be regulated by autocrine/paracrine mechanisms dependent upon locally produced CL hormones. The decreasing sensitivity of CL to exogenous PGF2 can be a mechanism for protection against premature luteolysis during the early to mid-luteal phase in cattle. Nevertheless, at the end of the luteal phase, PGF2 and OT may interact and activate luteal and non-luteal cells to initiate functional and morphological luteolysis [38, 46, 47]. Moreover, we suggest that the lack of response to PGF2 in early bovine CL is regulated on the receptor level and/or the postreceptor level and that it depends on locally produced hormones. In support of this suggestion, in ewes, an increase in mRNAs encoding protein kinase C inhibitors, and the associated increase in the corresponding proteins, may be involved in resistance of the CL to exogenous PGF2 during the early part of estrous cycle [48]. Therefore, multiple processes seem to be involved in regulating the responsiveness of PGF2 receptor-coupled protein G systems. One of these processes is homologous desensitization of PGF2 receptors in CL and may be due to long-lasting stimulation by PGF2 produced in the ovary. Moreover, both OT and P4 through their luteotropic actions on early to mid-CL may indirectly (via PGF2) or directly (via heterologous desensitization) affect the functionality of PGF2 receptors and/or formation of second messengers.
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ACKNOWLEDGMENTS |
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and PGE2; Dr. Per Melin of Ferring AB, Malmö, Sweden, for the OT antagonist; Dr. Krzysztof Chwalisz of Schering AG, Berlin, Germany, for the P4 antagonist; and the National Hormone and Pituitary Program, University of Maryland School of Medicine and the National Institute of Diabetes and Digestive and Kidney Disease (NIDDK), for bovine LH (USDA-bLH-B6). The authors also thank Dr. Timothy Marczylo for his assistance in preparing this manuscript and Mr. Shinya Kobayashi for his technical assistance. |
FOOTNOTES |
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2 Correspondence. FAX: 81 86 251 8388; kokuda{at}cc.okayama-u.ac.jp
3 Permanent address: Division of Reproductive Endocrinology and Pathophysiology, Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences, 10–718 Olsztyn-Kortowo, Poland.
Accepted: December 30, 1998.
Received: September 22, 1998.
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