Abstract
LH appears to be a potent stimulator of the release of endometrial prostaglandins (PGs) in the pig. The aim of the present studies was to examine the effect of LH on PGF2α and PGE2 secretion by cultured porcine endometrial cells on days 10–12 and 14–16 of the oestrous cycle and to compare its action with oxytocin. A time-dependent effect of LH (10 ng/ml) on PGF2α release from luminal epithelial and stromal cells on days 10–12 was observed (experiment 1). The highest increase in PGF2α secretion in response to LH was detected in stromal cells after 6 h of incubation (P < 0.001). Epithelial cells responded to LH after a longer exposure time (P < 0.01). A concentration-dependent effect of LH (0.1–100 ng/ml) on PGF2α release from stromal cells was examined after 6 h and from epithelial cells after 12 h (experiment 2). Effective concentrations of LH were 10 and 100 ng/ml. LH (10 ng/ml) and oxytocin (100 nmol/l) affected PGF2α and PGE2 secretion from endometrial cells on days 10–12 and 14–16 of the oestrous cycle (experiment 3). LH stimulated PGF2α secretion from both cell types and its action was more potent on days 10–12. LH induced PGE2 release, especially in epithelial cells on days 14–16. A stimulatory effect of oxytocin on PGF2α was confirmed in stromal cells, but this hormone was also shown to enhance PGE2 output. These results indicated that LH, like oxytocin, a very effective stimulator of PGF2α release, could play an important role in the induction of luteolysis.
Introduction
Pulsatile release of prostaglandin (PG) F2α from the uterus in the late luteal phase of the oestrous cycle induces corpus luteum regression in many species, including pigs (for review see McCracken et al. 1999). Oxytocin (OT) is the major known stimulus for increased PGF2α secretion at the time of luteolysis in pigs (Kieborz et al. 1991, Carnahan et al. 1996, Uzumcu et al. 1998). On the other hand, agreement between OT and 13,14-dihydro-15-keto-PGF2α (PGFM) peaks in the blood is only about 30% and blocking of OT receptors neither prevented luteolysis nor changed the duration of the oestrous cycle in gilts (Kotwica et al. 1999). This may indicate that OT is not mandatory to induce endometrial release of PGF2α at the time of luteolysis. Therefore, the induction and course of luteolysis is regulated not only by OT but apparently also by one or more other factors.
Porcine endometrium possesses functional luteinizing hormone (LH) receptors (Stepien & Ziecik 2002) and the incubation of endometrial strips with LH resulted in an increased PGFM accumulation in medium. The strongest effect was observed at the time of luteolysis when the numbers of LH receptors were also at their highest (Stepien et al. 1999). LH was shown to stimulate cyclooxygenase expression in the endometrium (Stepien et al. 1999) and increased PGF2α release (Ziecik et al. 2000) on days 14–16 of the oestrous cycle in pigs. Moreover, both systemic infusion (Ziecik et al. 2001) as well as intramuscular injection (Guthrie & Bolt 1983) of human chorionic gonadotrophin induced PGF2α secretion in vivo. This indicates that the endogenous LH pulses may provoke PG output from porcine endometrium and that LH participates in the process of luteolysis in this species.
The proper ratio between PGF2α and PGE2 seems to be responsible for the maintenance of corpora lutea and successful implantation (for review see Ziecik 2002). Despite many studies on the secretory properties of cultured porcine stromal and epithelial cells at different reproductive states (Zhang et al. 1991, Davis & Blair 1993) as well as cellular responsiveness to OT (Uzumcu et al. 1998, 2000) and steroids (Carnahan et al. 2002, Hu et al. 2003), little is known about the modulation of PGE2 output from endometrial cells in vitro. Our recent results revealed that secretion of both PGs depends on the day of the oestrous cycle and the type of cells as well as the time of incubation (Blitek & Ziecik 2004).
LH was shown to selectively affect the secretion of PGs from endometrial explants on days 14–16 of the oestrous cycle and early pregnancy, but it is not known which endometrial cell types are responsive to LH action around the time of luteolysis. The present studies were undertaken to examine the effect of LH on PGF2α and PGE2 secretion by cultured porcine stromal and luminal epithelial cells on days 10–12 and 14–16 of the oestrous cycle and to compare this with the action of OT.
Materials and Methods
Tissue
Uteri of 12 crossbred gilts (Large White × Polish Landrace) were obtained from a local abattoir within 20 min after exsanguination and transported in ice-cold phosphate-buffered saline (PBS) buffer (137 mmol/l NaCl, 27 mmol/l KCl, 10 mmol/l Na2HPO4 and 2 mmol/lKH2PO4, pH 7.4) to the laboratory within 1 h. The stages of the oestrous cycle were determined by macroscopic observation of the ovaries and uterus as previously described (Akins & Morissette 1968, Leiser et al. 1988). Endometrial tissue obtained on days 10–12 and 14–16 of the oestrous cycle was used. These two periods were selected because during days 10–12 luteolytic release of PGF2α is not yet observed but on days 14–16 the process of luteolysis is already initiated in cyclic gilts.
Isolation of endometrial cells
Porcine endometrial cells were isolated aseptically under a laminar flow hood using a procedure described recently (Blitek & Ziecik 2004). Briefly, a randomly selected uterine horn was washed with sterile PBS supplemented with 100 IU/ml penicillin and 100 μg/ml streptomycin (Sigma Chemical Co., St Louis, MO, USA) and cut longitudinally to expose the endometrium. Endometrial tissue was separated from the myometrium and digested with 0.48% (w/v) dispase (Gibco-BRL Life Technologies, Paisley, Strathclyde, UK) in Hanks’ balanced salt solution (HBSS), Ca-, Mg- and phenol red-free, pH 7.4; Sigma) at room temperature for 50 min with gentle shaking. Luminal epithelial cells released after this digestion were pelleted by centrifugation at 200 g for 10 min, washed once with Medium 199 (Sigma) containing 4% (w/v) bovine serum albumin (BSA; ICN Biomedicals, Inc., Costa Mesa, CA, USA), 100 IU/ml penicillin and 100 μg/ml streptomycin. Red blood cells were removed using red blood cell lysing buffer (Sigma). The luminal epithelial cells obtained were then washed three more times with fresh Medium 199 containing 4% BSA, resuspended in 3 ml culture medium (Medium 199 containing 2% BSA, 10% newborn calf serum (v/v; Sigma), and 100 IU/ml penicillin and 100 μg/ml streptomycin) and counted in a haemocytometer. The cell viability was higher than 90% as assessed by 0.5% (w/v) trypan blue dye exclusion
The remaining endometrial tissue was minced with scissors, placed in 0.25% trypsin solution (Biomed, Lublin, Poland) and digested for 1 h at 37 °C. The cell suspension was filtered to remove undigested tissue fragments, pelleted by centrifugation and washed with fresh medium. The remainder was further treated with 0.06% collagenase (w/v; Sigma) in Medium 199 containing 2% BSA for 1.5 h at 37 °C. The cell suspensions obtained after trypsin and collagenase digestions were pooled together, washed three times with fresh medium and counted in a haemocytometer. They were assessed as stromal cells and the cell viability was higher than 95%. The homogeneity of cells was evaluated by the immunofluorescent staining of cultured cells for cytokeratin and vimentin as described previously (Blitek & Ziecik 2004). The purity of stromal cells was 95–98% and luminal epithelial cells 85–90%.
Cells were cultured at 37 °C in a humidified atmosphere of 95% air:5% CO2. Stromal cells were washed 24 h after plating to remove contaminating epithelial cells before continuing culture. Cells were cultured for 64–88 h to allow them to adhere to the plates before initiation of experiments when monolayers were estimated to be approximately 90–95% confluent.
Experiment 1
To determine the time-course for PGF2α secretion in response to LH (pLH B-1; USDA Hormone Program, Bethesda, MD, USA) stromal and luminal epithelial cells obtained from six gilts at days 10–12 of the oestrous cycle were used. Cells were incubated with 0 or 10 ng/ml LH in Medium 199 (serum free) for 1, 3, 6, 12 or 24 h. At the end of each treatment period, media were collected and stored at −40 °C until PGF2α concentrations were estimated by enzyme immunoassay (EIA). Cells were lysed with 100 mmol/l NaOH and total cellular protein was measured (Bradford 1976).
Experiment 2
To study the dose-dependent PGF2α secretion in response to LH, stromal and luminal epithelial cells collected from six gilts (the same as in experiment 1) were treated with the following doses of LH: 0, 0.1, 1, 10 or 100 ng/ml. The time of incubation was chosen based on the results of experiment 1 and was 6 h for stromal cells and 12 h for epithelial cells. Cells were then lysed with 100 mmol/l NaOH and total cellular protein was measured (Bradford 1976).
Experiment 3
To study the effect of LH on PGF2α and PGE2 release, endometrial cells collected before (days 10–12) and at the time (days 14–16) of luteolysis were used. Time of incubation (6 h for stromal and 12 h for luminal epithelial cells) and the effective dose of LH (10 ng/ml) were chosen based on the results of experiments 1 and 2. Additionally, OT (Sigma) at the dose of 100 nmol/l (Uzumcu et al. 1998) was used as a positive control to study LH action on PGF2α release, but OT action on PGE2 secretion was examined for the first time and also compared with LH.
EIA of PGF2α and PGE2
Concentrations of PGF2α in incubation medium were determined by direct EIA test as described earlier (Uenoyama et al. 1997). Cross-reactivities of the anti-PGF2α serum (Sigma) were as follows: PGF1α 60%, PGE1 and PGE2 <0.1% and PGA1, PGA2, PGB1 and PGB2 < 0.01%. The PGF2α standard curve ranged from 0.11 to 30 ng/ml, and the median effective dose (ID50) was 2.59 ng/ml. The intra- and interassay coefficients of variation were 7.6 and 12.3% respectively.
Concentrations of PGE2 in incubation medium were determined by a direct EIA test as described earlier (Skarzynski & Okuda 2000). The anti-PGE2 serum was donated by Dr S Ito, Kansai Medical University, Osaka, Japan. Cross-reactivities of the anti-PGE2 serum were as follows: PGE1 18%, PGA1 10%, PGA2 4.6%, PGB2 6.7%, PGD2 0.13%, PGF2α 2.8%, PGJ2 1.4% and 15-keto PGE2 0.05%. The PGE2 standard curve ranged from 1.56 to 100 ng/ml and the ID50 of the assay was 11.2 ng/ml. The intra- and interassay coefficients of variation were 5.8 and 9.2% respectively.
Statistical analysis
The data are shown as the mean ±s.e.m. of values obtained in four to six separate experiments (pigs), each performed in duplicate. Levels of PGF2α and PGE2 production were standardized on protein concentrations per well (ng/mg protein). The statistical significance of differences between controls and treated groups was assessed by one-way ANOVA followed by Bonferroni multiple comparison tests (GraphPad PRISM version 4; GraphPad Software, Inc., San Diego, CA, USA).
Results
Experiment 1
LH stimulated PGF2α output (ng/mg protein) from both endometrial cell types in a time-dependent manner (Fig. 1). Basal and LH-stimulated secretion of luteolytic PG was tenfold higher for luminal epithelial than for stromal cells. However, the effect of LH was more visible in stromal than in epithelial cells. LH-stimulated increase of PGF2α secretion from stromal cells was observed during all time-periods studied (1–24 h). The most effective action of LH was detected after 6 h of incubation, when the amount of PGF2α released into medium increased almost twofold (36.2 ± 3.3 vs 69.7 ± 3.0; P < 0.001). Epithelial cells needed more time of LH treatment (12 and 24 h) to significantly increase PGF2α output (772.3 ± 21.2 vs 1452.9 ± 123.9 after 12 h and 3220.8 ± 187.0 vs 4326.9 ± 192.9 after 24 h; P < 0.01). No effect of LH on luminal epithelial cells was detected during the shorter periods of incubation (1–6 h).
Experiment 2
Periods of 6 h for stromal cells and 12 h for luminal epithelial cells were chosen to study the effect of different doses of LH (0.1–100 ng/ml) on PGF2α secretion on days 10–12 of the oestrous cycle. Both cell types revealed dose-dependent increase in PGF2α output after LH treatment (Fig. 2). Effective doses in the studied cells were 10 and 100 ng/ml (P < 0.01 and P < 0.001).
Experiment 3
Figure 3 shows the effect of LH (10 ng/ml) and OT (100 nmol/l) on PGF2α and PGE2 secretion from stromal cells after 6 h of incubation. Basal secretion of luteolytic PGF2α was almost threefold higher on days 14–16 than on days 10–12 of the oestrous cycle (103.04 ± 7.8 and 36.2 ± 3.3 respectively; P < 0.001). On the other hand, non-stimulated release of luteotropic PGE2 was comparable in both periods of the oestrous cycle studied. Both hormones stimulated (P < 0.001) PGF2α output from cells collected on days 10–12 of the oestrous cycle. The response of cells obtained on days 14–16 was greater to OT (P < 0.001) than to LH (P < 0.05) treatment. LH- as well as OT-stimulated increase in PGE2 output was observed in cells collected on days 14–16 (P < 0.05). However, only OT affected PGE2 secretion (P < 0.05) from stromal cells on days 10–12 of the oestrous cycle.
Modulation of PGF2α and PGE2 secretion (ng/mg protein) from luminal epithelial cells by LH and OT is shown in Fig. 4. The time of incubation was extended to 12 h based on the results of experiment 2. Basal release of PGF2α was higher from cells obtained on days 10–12 than on days 14–16 (P < 0.001). Both LH and OT increased PGF2α secretion on days 10–12 (P < 0.01 and P < 0.05 respectively), but only OT had a stimulating effect on days 14–16 (P < 0.01). The output of PGE2 from luminal epithelial cells increased in response to LH and OT significantly in both periods of the oestrous cycle studied. The more visible effect of LH on PGE2 release was found in epithelial cells collected on days 14–16 (P < 0.01) than on days 10–12 (P < 0.05) of the oestrous cycle.
Discussion
One of the intriguing questions about luteolysis is what signals initiate the production and release of PGF2α from the endometrium. In pigs, increased concentrations of PGF2α in utero-ovarian venous plasma was observed on days 12–13 of the oestrous cycle (Gleeson et al. 1974, Moeljono et al. 1977). If LH participates in the initiation of luteolysis by increasing PGF2α secretion from the endometrium it should exert its action especially around day 12. Cells collected later in the oestrous cycle (days 14–16) already received a luteolytic signal and LH could act as a modulator of PG production.
In our studies OT served as a positive control for studying stimulated PGF2α secretion (Uzumcu et al. 1998, 2000), but its effect on PGE2 release from cultured porcine endometrial cells has been here examined for the first time. The present study demonstrated that, in pigs, LH stimulated PGF2α secretion from cultured endometrial stromal and luminal epithelial cells. The action of LH depended on the cell type and the day of the oestrous cycle as well as the duration of incubation with the hormone. The results of our experiments also revealed a different cellular response to LH. Stromal cells were more sensitive to LH, and LH-stimulated increase in PGF2α secretion was observed after all periods of incubation studied (1–24 h). Luminal epithelial cells responded with elevated PG output after longer (12 and 24 h) hormone treatment periods. The present results were not expected since LH receptor mRNA and protein expression were present mainly in the epithelium of porcine endometrium (B Gawronska, A Stepien & A J Ziecik, unpublished observations). Our previous studies (Stepien & Ziecik 2002) showed that endometrial LH receptors are functional because treatment of isolated cells (a mixture of luminal epithelial cells with some stromal and glandular epithelial cells) resulted in higher cAMP output and increased accumulation of inositol phosphates in medium. In addition to PG production, LH could be also responsible for other activities, such as protein secretion in these cells. Nevertheless, further studies on the number of LH receptors and second messenger systems in separated and cultured porcine stromal and epithelial cells are needed to clarify the different cellular response to this hormone.
Similar responsiveness of cultured porcine endometrial cells was previously observed during OT treatment. Stromal cells were the most responsive and luminal epithelial cells the least responsive to this hormone (Uzumcu et al. 1998), even though OT receptor expression appeared to be greater within the epithelium than stroma of the porcine endometrium (Boulton et al. 1995). Further studies on polarized luminal epithelial cells revealed that these cells were completely unresponsive to a 3-h long treatment with exogenous OT (Braileanu et al. 2000). The authors of those studies concluded that epithelial cells may not contribute to OT-stimulated pulses of PGF2α. However, the results of Uzumcu et al.(1998) indicated that after longer incubation (12 h), OT showed a tendency (no statistical differences) to increase PG secretion. These data are in agreement with our results since longer incubation resulted in elevated PGF2α release in response to both OT and LH. On the other hand, OT is a peptide with a very short half-life, and the stimulatory effect of this hormone observed after 12 h may not be due to its direct effect on PG synthesis. However, cultured bovine endometrial epithelial cells, which are the main target of OT action (Asselin et al. 1996), responded in increased PGF2α production after 12 and 24 h (Parent et al. 2003).
In the present study, LH action on PGF2α secretion was more effective in cells collected before the onset of luteolysis. An almost twofold increase in PGF2α concentration in the medium was observed in stromal and luminal epithelial cells obtained on days 10–12 of the oestrous cycle. These findings support our observations on the possible participation of LH in the PG surge at luteolysis. LH is released in a pulsatile manner during the luteal phase of the oestrous cycle (Parvizi et al. 1976, Van de Wiel et al. 1981). The porcine uterus could be exposed in vivo to the dose of LH used in our experiment (10 ng/ml), since the highest pulses of LH occurring in the mid-luteal phase of the oestrous cycle and at the time of luteolysis (days 13–16) vary from 5 to 10 ng/ml (Van de Wiel et al. 1981, Ziecik et al. 2001). Moreover, much greater agreement was found between LH and PGFM (79.2%; Ziecik et al. 2001) than between OT and PGFM peaks (30%; Kotwica et al. 1999). However, in the present study, the influence of OT on PGF2α output was highly effective especially in stromal cells, which were more sensitive to OT than to LH action. Such a result could be caused by a combined effect of exogenous and endogenous OT. It was shown that OT is produced by porcine stromal and epithelial cells and may act in an auto- and/or paracrine manner to increase PGF2α output (Hu et al. 2001). Additionally, in our studies, OT like LH, was more effective before luteolysis (days 10–12). These findings are in agreement with earlier data (Uzumcu et al. 2000), in which stromal cells collected on day 12 responded to OT to a higher degree than on day 16 of the oestrous cycle. Since stromal cells are present in the uterus in much greater number than luminal epithelial cells (Blackwell et al. 2003) they are expected to contribute the major portion to endocrine secretions of PGF2α. Moreover, their proximity to the uterine vasculature also predestines them to be the main source of luteolytic levels of PGF2α. Consequently, OT as well as LH could be important stimulators for PGF2α release from the uterus at the time of luteolysis.
In the present studies, basal secretion of PGE2 was comparable on days 10–12 and 14–16 in stromal as well as in epithelial cells. PGE2 release from the endometrium was increased by both LH and OT on all the days of the oestrous cycle studied, especially in epithelial cells. On days 14–16, PGF2α secretion was only slightly increased after treatment of these cells with LH but in the same period the output of PGE2 was substantially increased. LH preferentially stimulated PGF2α compared with PGE2 in epithelial cells obtained on days 10–12, whereas OT highly increased PGE2 output. Bovine endometrial epithelial cells treated with OT for 24 h also responded with elevated PGF2α (14-fold) and PGE2 (fourfold) concentrations in the incubation medium (Parent et al. 2003). PGF2α and PGE2 are synthesized directly from endoperoxide H2 by PGF synthase (PGFS) and PGE synthase (PGES) respectively. The basal and stimulated PG secretion by endometrial cells in vitro is dependent on different patterns of PGFS and PGES expression on mRNA and protein levels (Waclawik et al. 2004). Another source of PGF2α is PGE2, which could be converted into PGF2α by PGE2 9-keto reductase. Despite the great significance of PGE2 9-keto reductase as a regulator the of PGE2/PGF2α ratio, little is known about the modulation of this enzyme expression. The activity of PGE2 9-keto reductase was inhibited by oestradiol and progesterone but stimulated by OT and calcium ions in human decidua vera (Schlegel et al. 1984). Moreover, oestradiol strongly inhibited PGE2 9-keto reductase in the bovine placenta (Kankofer & Wiercinski 1999). Thus, during the maternal recognition of pregnancy in pigs, oestrogen of conceptus origin can increase the PGE2/PGF2α ratio which is essential for luteal maintenance. On the other hand, the LH-stimulated release of PGE2 in cyclic gilts found in our studies would not prevent luteolysis, since PGE2 could be converted to PGF2α (Okrasa et al. 1985) and the number of PGE2 receptors on porcine luteal cells is decreased on days 12–14 of the oestrous cycle (Feng & Almond 1999).
In the present studies, basal PGF2α release from stromal cells was increased on days 14–16 in comparison with days 10–12, but decreased about twofold in epithelial cells on comparable days. The same pattern of non-stimulated PGF2α secretion from cultured porcine endometrial cells on days 12 and 16 has been demonstrated previously (Uzumcu et al. 2000). It has been well documented that porcine stromal cells principally produce PGE2 and epithelial cells produce more PGF2α (Zhang et al. 1991, Blitek & Ziecik 2004). However, our previous results (Blitek & Ziecik 2004) revealed that, in stromal cells, the ratio between PGF2α and PGE2 depends on the time of incubation. Long incubation (24 h) resulted in balanced production of both PGs or even a tendency for the release of PGF2α to dominate. Basal PGF2α secretion from epithelial cells was approximately tenfold greater than from stromal cells. This may be caused by the higher expression of cyclooxygenase COX-2 (a key enzyme in PG synthesis), but also by a greater availability of the substrate, arachidonic acid, in epithelial cells. However, this suggestion needs detailed investigations.
In summary, these results revealed that stromal cells were more sensitive to LH than epithelial cells. Furthermore, LH action was more effective in stimulating PGF2α secretion from cells collected before luteolysis. However, PGE2 release was also increased in the presence of LH, especially in epithelial cells obtained during luteolysis. Additionally, our studies confirmed the stimulatory effect of OT on PGF2α secretion from stromal cells of the porcine endometrium, but this hormone was also shown to enhance PGE2 output. Moreover, OT seems to be a more potent modulator of PG secretion from the endometrium of the pig in in vitro conditions. The overall results indicate that LH and OT could play an important role in the induction of luteolysis, since they are very effective stimulators of PGF2α release from endometrial cells obtained on days 10–12 of the oestrous cycle. The results of the present and earlier studies (Uzumcu et al. 1998, 2000, Stepien et al. 1999, Ziecik et al. 2000, 2001, Carnahan et al. 2002, Stepien & Ziecik 2002, Hu et al. 2003) on endometrial cells indicate that, in pigs, PG production at the time of luteolysis is regulated by at least two factors (i.e. OT and LH). Until now it is still not clear which of them dominates.
Time-dependent effect of LH (10 ng/ml) on PGF2α output by cultured porcine (A) stromal and (B) luminal epithelial cells obtained at days 10–12 of the oestrous cycle. Data are presented as means ±s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001.
Citation: Reproduction 130, 1; 10.1530/rep.1.00639
Dose-dependent effect of LH on PGF2α output by cultured porcine (A) stromal and (B) epithelial cells obtained at days 10–12 of the oestrous cycle. LH (0.1–100 ng/ml) was added for 6 h in stromal and 12 h in luminal epithelial cell cultures. Data are presented as means ±s.e.m. **P < 0.01, ***P < 0.001 when compared with control value.
Citation: Reproduction 130, 1; 10.1530/rep.1.00639
Effect of LH (10 ng/ml) and OT (100 nmol/l) on PGF2α and PGE2 secretion by cultured porcine stromal cells obtained on days 10–12 and 14–16 of the oestrous cycle. Cells were incubated with hormones for 6 h. Data are presented as means ±s.e.m. *P < 0.05, ***P < 0.001 when compared with control value.
Citation: Reproduction 130, 1; 10.1530/rep.1.00639
Effect of LH (10 ng/ml) and OT (100 nmol/l) on PGF2α and PGE2 secretion by cultured porcine luminal epithelial cells obtained on days 10–12 and 14–16 of the oestrous cycle. Cells were incubated with hormones for 12 h. Data are presented as means ±s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 when compared with control value.
Citation: Reproduction 130, 1; 10.1530/rep.1.00639
We thank Dr S Ito of Kansai Medical University for the PGE2 antiserum. The authors are grateful to Ms K Gromadzka-Hliwa and Mr J Klos for technical help with the PG EIAs. This research was supported by the State Committee for Scientific Research as a solicited project PBZ-KBN-084/P06/2002 from 2003 to 2005. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.
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