Molecular Human Reproduction, Vol. 6, No. 8, 681-687,
August 2000
© 2000 European Society of Human Reproduction and Embryology
Endocrinology |
Nitric oxide induces apoptosis in the human corpus luteum in vitro
1 Institute of Maternal and Child Research and 2 Pathology Department, San Borja–Arriaran Clinical Hospital, School of Medicine, University of Chile
Abstract
The present study aimed to investigate the role of nitric oxide (NO) in regression of the human corpus luteum. We therefore examined the effect of both NO and human chorionic gonadotrophin (HCG) on luteal cell apoptosis, and Bcl-2 production. The effect of NO on oestrogen production during corpus luteum regression was also studied. Slices from corpus luteum collected throughout the luteal phase were incubated for 4 h with the nitric oxide synthase (NOS) substrate, L-arginine (L-Arg, 1 mmol/l), the NOS inhibitor N-monomethyl-L-arginine (L-NMMA) (1 mmol/l), or with HCG (10 IU/ml). Oestradiol concentrations were determined by radioimmunoassay; Bcl-2 concentrations were measured by enzyme-linked immunosorbent assay; apoptosis was detected in-situ by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling; and inducible nitric oxide synthase (iNOS) was assessed by immunohistochemistry. Consistent with our previous findings, L-Arg elicited an inhibitory action on the production of oestradiol (P < 0.05). The number of apoptotic cells increased (P < 0.05) from early to late corpus luteum, as did the number of cells positive for the expression of iNOS. The percentage of apoptotic cells in mid and late luteal phase was increased by L-Arg (56% and 310% respectively; P < 0.05), and decreased by L-NMMA and HCG. Although no changes were observed in Bcl-2 concentration during the corpus luteum life span, L-Arg inhibited, and HCG augmented, Bcl-2 production (P L 0.05) from mid and late corpus luteum cells in vitro. In summary, these results suggest that the opposite actions of L-Arg and HCG on human corpus luteum viability may, in part, be mediated by changes in the level of the anti-apoptotic activities caused by oestradiol and Bcl-2 protein.
apoptosis/Bcl-2/human corpus luteum/nitric oxide
Introduction
Luteal regression is necessary for the cyclicity of the reproductive process, and occurs as the synchronous loss of cellular function and subsequent cell death within the corpus luteum. Recent reports have suggested that luteal regression or luteolysis in many species (Zelesnik et al., 1989
; Juengel et al., 1993; Dharmarajan et al., 1994; Zheng et al., 1994; Rueda et al., 1995), including the human (Shikone et al., 1996), may represent a form of programmed cell death or apoptosis, a highly conserved form of physiological cell death important for tissue development and homeostasis. The apoptotic programme is characterized by morphological and biochemical changes within the cells. The most striking feature of apoptosis is the activation of endonuclease activity, and internucleosomal DNA cleavage has been described in luteal cells of regressing human corpus luteum (Shikone et al., 1996).
Several molecules such as cytokines, growth factors, peptide hormones, reactive oxygen species (ROS) (Feuerstein, 1999) and steroids (Billig et al., 1993; Evans-Storm and Cidlowski, 1995) have been reported to regulate apoptosis in different tissues. It is well documented that the induction of apoptosis occurs by addition or withdrawal of steroid hormones from target tissues. In fact, withdrawal of androgens induces apoptosis of ventral prostate epithelial cells with the concomitant regression of the prostate, whereas progestins are important compounds for the maintenance of the lactating breast tissue (Tenniswood et al., 1992) and for uterine epithelial cell survival (Rotello et al., 1992). Furthermore, oestrogens inhibit and androgens enhance ovarian granulosa cell apoptosis (Billig et al., 1993). On the other hand, nitric oxide (NO) has been suggested to have a functional role in the regulation of the corpus luteum. In this regard, an oxidative stress condition occurs in the mid stage human corpus luteum (Vega et al., 1994a), concomitant with the expression of endothelial NO synthase (NOS) mRNA and protein (Vega et al., 1998). Furthermore the inhibitory effect of NO on oestradiol production by the human corpus luteum may be due in part to a decrease in P450 aromatase (P450arom) activity (Van Voorhis et al., 1994; Olson et al., 1996; Johnson et al., 1999; Kagabu et al., 1999). On the other hand, many studies have shown that NO has both cytotoxic and cytoprotective effects. The proapoptotic effects appear to be linked to the production of high concentrations of NO by the activity of inducible NOS (iNOS). The anti-apoptotic effects are mainly mediated by low amounts of NO or stimulation of endothelial NOS (Knowles and Moncada, 1994; Dimmeler and Zeiher, 1997).
Regardless of the fact that the inducers or inhibitors of programmed cell death may differ in different species or cell types, the basic molecular and cellular events underlying apoptosis may involve changes in the expression of the same genes. In fact, cell death is controlled by the expression of ubiquitous factors such as c-myc, p53 and members of the Bcl-2 family, among others. Therefore, it is likely that the relative ratios of Bcl-2, which protects against cell death, and Bax, a 21 kDa proapoptotic protein related to Bcl-2, will determine the fate of a cell rather than the absolute concentrations. In relation to the human corpus luteum, some immunohistochemical studies have demonstrated that the level of expression for the product of the proto-oncogene Bcl-2 remains unchanged throughout the luteal phase (Rodger et al., 1995), whereas in the bovine corpus luteum elevated levels of Bax has been associated with structural luteolysis (Rueda et al., 1997). The mechanism by which these genes regulate apoptosis remains to be fully identified; however, it has been suggested that Bcl-2 and related proteins may regulate levels of reactive oxygen species (ROS) or their intermediates as one possible mechanism to control apoptosis (Dharmarajan et al., 1999). To increase our understanding of the mechanisms causing apoptosis in human corpus luteum regression, we have investigated the effect of both NO and human chorionic gonadotrophin (HCG) on the incidence of apoptosis and the production of the anti-apoptotic protein, Bcl-2, in corpus luteal cells in vitro.
Materials and methods
All chemicals, incubation media, and hormones used were obtained from Sigma Chemical Co. (St Louis, MO, USA), except for N-monomethyl-L-arginine (L-NMMA) (Calbiochem Corp., La Jolla, CA, USA), the apoptosis detection fluorescein kit (Promega, Madison, WI, USA), or monoclonal antibody against iNOS isoform (mouse anti-mouse iNOS, N32020, Clone 6; Transduction Laboratories, Lexington, KY, USA), the enzyme-linked immunosorbent assay (ELISA) kit (Endogen, Woburn, MA, USA) for measurement of Bcl-2 concentrations. Primers, DNase I (Amp Grade), TRIzol, SuperScript RT II, BioNick Labeling System and Taq DNA Polymerase were purchased from GibcoBRL Life Technology (Bethesda, MD, USA).
Subjects
Corpora lutea were obtained from 20 eumenorrhoeic women, aged 27–40 years, undergoing laparotomy for tubal sterilization (n = 14) or myomas (n = 6) at the San Borja–Arriaran Clinical Hospital, University of Chile, National Health Service (Santiago, Chile). None of the patients had experienced infertility or endometriosis and had stopped any hormonal contraception at least 3 months before surgery. The study was approved by the Institutional Review Board and informed written consent was obtained from all patients before surgery. After removal of the corpus luteum, the tissue was placed in a sterile phosphate-buffered saline solution (PBS) and transported immediately to the laboratory at room temperature. The cycle-date day of each woman was confirmed by an endometrial biopsy (Noyes et al., 1950) and classified as early (1–4 days; n = 6), mid (5–9 days; n = 8), or late (10–14 days; n = 6) luteal phase. The tissue was then cut into slices, with weights ranging between 20 and 70 mg (wet weight). One piece of each corpus luteum was placed in 4% formalin/PBS (pH 7.2–7.4) for histological evaluation (Corner, 1956), cell death determination and immunohistochemical study, and another piece was frozen in liquid nitrogen after weighing and stored at –70°C for later determination of Bcl-2 concentration or mRNA preparation (control condition; time 0, no incubation).
Incubation procedures and oestradiol radioimmunoassays
For experiments assessing the influence of NO on cell survival, tissue slices were incubated as previously described with some modifications (Vega et al., 1987). Briefly, the slices were placed into incubation plates with Hanks' (L-Arg-free) media supplemented with glutamine (0.1 mg/ml), bovine seum albumin (0.1% w/v), NaHCO3 (26 mmol/l), HEPES (25 mmol/l), antibiotics (100 IU/ml penicillin and 5 mg/ml streptomycin), pH 7.4, to give a final volume of 1.0 ml. Basal and treated conditions were carried out in duplicates or triplicates. HCG (10 IU/ml), the NOS substrate L-Arg (1.0 mmol/l), or the competitive inhibitor of NOS, L-NMMA (1.0 mmol/l) were then added to some plates. Incubations were terminated at 4 h and the media were stored at –20°C until assayed for oestradiol by specific radioimmunoassay as reported earlier (Vega et al., 1994b). The intra-assay coefficient of variation for the oestradiol radioimmunoassay was 4.1% and the inter-assay coefficient of variation was 6.7%; the sensitivity was 5 pg/tube and the cross-reactivity of oestradiol and oestrone was <0.02, being <0.01% with other steroids. After incubation, one tissue slice was frozen for later determination of Bcl-2 concentration or mRNA preparation and another piece was fixed in formalin/PBS and embedded in paraffin wax for determination of apoptosis by the TUNEL method (von Rango et al., 1998).
In-situ detection of apoptosis by TUNEL
Nuclear DNA fragmentation was assessed in 4–6 µm thick sections of corpus luteum mounted on silane-coated slides, deparaffinized with xylene and rehydrated, by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling method (TUNEL) using the apoptosis detection fluorescein kit and according to the specifications of the manufacturer. The method was adapted to the conditions required for use in the human corpus luteum. We used only nuclease-free solutions during the whole procedure, to avoid artificial DNA fragmentation. Slides were analysed by fluorescence microscopy with a wide band excitation barrier filter suitable for analysing both green (fluorescein-labelled fragmented DNA) and red (propidium iodide counterstain identifying cell nucleus) fluorescence. Three independent observers counted apoptotic cells in corpus luteum. The different optical fields (magnification x400) were selected in a random manner counting at least 3000 cells for each sample. Positive control tissues were corpus luteum samples pretreated with DNase (5 µg/ml, type I). To estimate non-specific binding and autofluorescence, negative controls (sections treated without the TdT enzyme) were included in all assays. No necrosis was observed as assessed by morphological criteria.
Immunohistochemical detection of iNOS isoform
The detection of inducible NOS (iNOS) was carried out as reported earlier (Vega et al., 1998) in histological paraffin wax sections (4–6 µm) of human corpus luteum of different ages. The monoclonal antibody was raised in mice against a 21 kDa protein fragment corresponding to amino acids 911–1144 of mouse iNOS (dilution 1:200) and has complete cross-reaction with human iNOS. The streptavidin–biotin peroxidase system with diaminobenzidine as the chromogen was used, and cell nuclei were stained with haematoxylin.
RNA preparation, cDNA synthesis and reverse transcription–polymerase chain reaction (RT–PCR) assessment
Total RNA was prepared from frozen corpus luteum of different stages (control condition) or from mid corpus luteum incubated in the absence (basal condition) or in the presence of 1 mmol/l L-Arg as was described previously (Johnson et al., 1997). The method of cDNA synthesis has also been previously described (Johnson et al., 1997). A PCR fragment of 672 bp was generated with primers based on sequences 702–722 (upstream: 5'-AGC TCT CCT CAT CAA ACC AGA-3') and 1354–1374 downstream (5'-GGC TTT CAT CAT CAC CAT GGC-3') of a human placental P450arom cDNA (Mahendroo et al., 1991). The reaction condition has been previously described (Johnson et al., 1997). For an internal control, a fragment of 661 bp of ß-actin was amplified in the same conditions as described above. Both P450arom and ß-actin cDNA amplifications were repeated for 30 cycles and were within the linear range. The levels of P450arom and ß-actin were evaluated by computerized analysis of optical density of the gel using an NIH Image version 1.61. To verify that the PCR-generated bands were P450arom cDNA, PCR products were resolved in 1% agarose gel, transferred and immobilized on neutral nylon membrane and hybridized at 42°C for 18 h with a human P450arom probe biotinylated using the BioNick Labeling System. The detection process was performed as indicated in the PhotoGene System 2.0 manual.
Determination of Bcl-2 concentration
Tissue samples were disrupted at 4°C in extraction buffer (1% Triton X-100; 2 mmol/l, 20 µg/ml aprotinin; 20 µg/ml leupeptin in 20 mmol/l HEPES, pH 7.4). Each extract was centrifuged at 14 000 g for 5 min and protein concentration was determined by the Microprotein-PR Sigma Diagnostic Assay. Bcl-2 concentration was evaluated by ELISA at 450 nm using a commercial kit. The sensitivity of the assay was <5 units/ml with an intra- and inter-assay variation coefficient <10%, and the results were expressed as units/mg protein.
Statistical analysis
The data are presented as mean ± SEM as indicated in the figure legends. The results were analysed using the non-parametric tests of Kruskall–Wallis and Wilcoxon for the number of cells/unit area. For comparison between basal and stimulated conditions for steroid production, expression studies and Bcl-2 concentrations, an unpaired Student's t-test was used. P < 0.05 was considered statistically significant.
Results
Effect of nitric oxide on oestradiol production
To confirm the action of NO on oestradiol production, human corpus luteum slices were incubated with the NOS substrate, L-Arg. Although dependent on the age of the corpus luteum, an inhibitory effect in oestradiol production was observed (Figure 1), consistent with our previous results in luteal cell cultures (Vega et al., 1998; Johnson et al., 1999). In fact, the addition of L-Arg caused a significant decrease (P < 0.05) in oestradiol production, as compared to corresponding basal values in mid (35%) and late (48%) corpus luteum, whereas no significant modification was observed in early corpus luteum (Figure 1). To study a possible action of endogenous NO, the NOS inhibitor L-NMMA was added to some cultures, and oestradiol production in mid and late corpus luteum was significantly enhanced (P < 0.05) by 30% and 36%, respectively (Figure 1), as compared to basal values.
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Effect of nitric oxide on P450arom expression
The presence of specific P450arom mRNA was detected by RT–PCR in human corpus luteum of different ages and in mid corpus luteum incubated without (basal) or with L-Arg for 4 h, as observed in the representative gel shown in the insert of Figure 2A and B
. The resulting 671 and 661 bp amplification products were the size predicted based on the cDNA sequence of P450arom and ß-actin, respectively. As observed in Figure 2A, P450arom expression, normalized to ß-actin expression, exhibited a decreased profile throughout the luteal phase. In fact, gene expression was significantly diminished (P < 0.05; 70%) in late relative to early human corpus luteum. To assess the action of NO on P450arom gene expression, L-Arg was added to the incubation media of mid corpus luteum slices. The results show a significant decrease (P < 0.05; ~25%) of P450arom expression in the tissue (Figure 2B).
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In situ detection of apoptosis
The TUNEL method was used to detect the fragmentation of the DNA which is one of the first morphological changes of the apoptotic process. As shown in Figure 3
, the percentage of cells with apoptotic positive signals increased in the control condition from early to late corpus luteum (P < 0.05; early, 0.5 ± 0.12%; mid, 1.60 ± 0.49%; late, 5.77 ± 1.53%), and no differences were observed after 4 h of incubation (basal condition). In addition, the presence of L-Arg in the media induced a significant increase in the percentage of apoptotic cells found in mid (56%) and late (310%) tissue slices (P < 0.05) (Figure 4), but not in early corpus luteum.
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To examine the effect of endogenous NO on the apoptotic process, L-NMMA was added to some cultures (Figure 4
). The results show a 32% decrease in the percentage of apoptotic cells found in late corpus luteum slices, as compared to basal values (P < 0.05). Furthermore, apoptosis was significantly diminished by the action of HCG, particularly in late tissue slices (40% decrease; P < 0.05), when compared to corresponding basal values.
Interestingly, the percentage of cells positive for the expression of iNOS isoform (Figure 5) was also increased in the late (4.7 ± 0.3%) compared to the early (0.3 ± 0.02%) and mid (2.2 ± 0.3%) corpus luteum (P < 0.05).
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Bcl-2 concentration in human corpus luteum
The data in Table I
represent basal Bcl-2 concentration found in corpus luteum during its life span. Regardless of the age of the corpus luteum, no changes were observed in the concentration of Bcl-2. Nevertheless, exogenous L-Arg (Figure 6) induced a significant decrease in Bcl-2 concentration in mid (30%; P < 0.05) and late (18%; P < 0.05) tissue, as compared to basal values. Meanwhile, a non-significant decrease in Bcl-2 concentration was observed in the early stage. On the other hand, an increased amount of Bcl-2 was observed in mid (43%) and late (51%) corpus luteum in the presence of HCG (P < 0.05) (Figure 6), indicating an important anti-apoptotic action of the gonadotrophin during human corpus luteum regression.
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Discussion
Nitric oxide is one of the compounds produced by human luteal cells, and in this regard we have previously reported that the expression of iNOS, which is the more active NOS isoform, is apparently restricted to a few non-steroidogenic luteal cells, whereas the constitutive endothelial NOS isoform is largely expressed in steroidogenic and endothelial cells within the human corpus luteum (Vega et al., 1998) in agreement with a recent study (Friden et al., 2000). These findings are consistent with other studies showing that endothelial NOS is present in human granulosa cells (Van Voorhis et al., 1994) and also in rat luteinized ovaries (Olson et al., 1996). Moreover, NO promotes luteolysis by eliciting an antisteroidogenic action in the human corpus luteum (Vega et al., 1998; Johnson et al., 1999), similar to that observed in cultured ovarian granulosa cells of human (Van Voorhis et al., 1994), rat (Olson et al., 1996), and porcine (Masuda et al., 1997) and confirmed by the results of the present investigation. The decrease in P450arom activity induced by NO in human granulosa cells and recently reported for the human corpus luteum (Johnson et al., 1999) may be attributable to the down-regulation of aromatase gene transcription observed in cultured human granulosa cells (Kagabu et al., 1999). This is in agreement with the results obtained in the present study, which showed that L-Arg reduced aromatase gene expression in human corpus luteum. In this regard, gonadal steroids have been reported to elicit an anti-apoptotic effect in ovarian cell types, such as granulosa cells, probably through regulation of the activity of calcium- and magnesium-dependent endonuclease. In fact, oestrogens inhibited whereas androgens enhanced DNA fragmentation in rat granulosa cells, whereas progesterone did not affect ovarian cell death (Billig et al., 1993). It is not known whether oestradiol exhibits an anti-apoptotic action in the human corpus luteum and this needs further investigation.
In previous studies, it has been demonstrated that cell death during luteal regression is associated with apoptotic DNA patterns. In fact, by using human corpus luteum obtained throughout the menstrual cycle and early pregnancy and processed for biochemical analysis of DNA integrity, a significant increase of apoptotic DNA cleavage in late human corpus luteum has been demonstrated (Shikone et al., 1996). These results are in agreement with the data obtained in the present investigation which clearly show, as evidenced by the in-situ detection using the TUNEL method, that the number of luteal cells undergoing apoptosis increases throughout the life span of the corpus luteum. Apoptosis is well known as a sequence of cellular events regulated by several proteins (caspases, endonucleases, among others), with every pathway converging in fragmentation of DNA. Therefore, the TUNEL method used in the present investigation, as in other studies (Gravieli et al., 1992; von Rango et al., 1998), is the best way to show apoptotic activity in histological sections and confirmed by morphological criteria. Nevertheless, the present study does not determine whether DNA fragmentation is present predominantly in a particular cell type within the corpus luteum, in large or small steroid-secreting cells and/or non-steroid secreting cells. It will be of importance to determine the degree of apotosis in the different luteal cell subpopulations, since steroid production, although diminished, continues to occur during corpus luteum regression.
Furthermore, our results indicate that L-Arg causes an increase in cell death from the early to the late stage of the human corpus luteum, exhibiting the same profile as the change in number of iNOS positive cells in this tissue. This observation is particularly important since it is known that iNOS activity is responsible for the production of large amounts of NO, which in turn may be linked to the proapoptotic effects described in other tissues (Dimmeler and Zeiher, 1997). In this respect, previous findings from our group have suggested that iNOS is the isoform that mainly generates NO in cultured human luteal cells (Johnson et al., 1999).
In the present study, the inhibition of endogenous NO generation by the specific NOS inhibitor resulted in a significant decrease in the number of cells with apoptotic positive signs. Since NO can be associated with ROS, these results would support the proposed action of oxidative damage on cell viability. On the other hand, apoptosis was significantly reduced in the HCG-stimulated condition, particularly in the late corpus luteum, indicating that the principal luteotrophic hormone may constitute an important factor for luteal rescue and maintenance through its action in the corpus luteum. In this regard, we have previously reported (Vega et al., 1994a) that HCG may cause a marked increase in the activity of superoxide dismutase (SOD) in the late corpus luteum, providing an enhancement in antioxidant protection. In agreement with these findings, it has been shown that SOD expression is significantly increased in isolated rabbit corpus luteum by treatment with HCG (Dharmarajan et al., 1999). In addition, we have shown that oestradiol, known to exhibit an antioxidant activity greater than that of vitamin E (Nakano et al., 1987), significantly decreased the lipid peroxidative index in human luteal cell cultures (Vega et al., 1994a).
Besides HCG, the many other regulatory molecules that play a role in protecting cells from apoptosis includes the Bcl-2 proto-oncogene product. As reported earlier (Hockenbery et al., 1990; Korsmeyer et al., 1995), Bcl-2 localization in mitochondrial membranes may be linked to the partial reduction of ROS accumulation, and thus a protection to a variety of cell types, from an anti-apoptotic effect (Kane et al., 1993). Our data indicate that Bcl-2 production is unchanged in the human corpus luteum throughout the luteal phase in agreement with previous observations in human corpus luteum (Rodger et al., 1995) in which Bcl-2 was determined by immunohistochemistry and immunoblotting. To our knowledge, the present investigation represents the first study to report a quantitative evaluation of the protein Bcl-2 in human corpus luteum during its life span. Also, it reveals that HCG has a protective role on human corpus luteum viability, especially in mid and late corpus luteum, in part, by the stimulation of Bcl-2 production. On the other hand, L-Arg treatment induces an age-dependent decrease in steroid production and in the anti-apoptotic Bcl-2 product, concomitantly with an increase in the number of cells presenting DNA fragmentation. It is not known whether the action of either NO or HCG on Bcl-2 production is on Bcl-2 synthesis or on its degradation or on both processes. Collectively, these results suggest that the NO-induced apoptosis in human corpus luteum may be partially reverted by the protective action of HCG during luteal cell regression. This potential anti-apoptotic effect of the gonadotrophin may be mediated in part by an increase in SOD activity (as mentioned earlier) and by the enhancement of Bcl-2 production in human corpus luteum.
In conclusion, the proapoptotic effect of NO in the corpus luteum may be mediated by the decrease of Bcl-2 expression, as well as by the differential regulation of other factors, such as Bax, or other compounds not yet identified but generated in the human corpus luteum during its life span. Furthermore, it is important to consider that an adequate corpus luteum structure and function will determine a normal endometrium, thus abnormal cell death of the corpus luteum may be relevant in evaluating recurrent pregnancy loss of unknown aetiology.
Acknowledgments
The authors are grateful to Andrea Castro (School of Medicine, University of Chile) for helpful comments on the manuscript; Alberto Palomino and Armando Cortinez (National Health Service, Chile) for their role in recruitment and surgical procedures of subjects; and Alejandra Villavicencio (Postgraduate PLACIRH Fellow, University of Chile) for helping with Bcl-2 determination. We also wish to thank Evan Simpson (Center for Reproductive Biology Sciences, University of Texas, Southwestern Medical Center at Dallas, USA) for the generous gift of P450arom probe; and the women who donated tissue. This work was supported by FONDECYT 1980899.
Notes
3 To whom correspondence should be addressed at P.O.Box 226–3, IDIMI, University of Chile, Santiago, Chile. E-mail: mvega{at}machi.med.uchile.cl 
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Submitted on January 31, 2000; accepted on May 22, 2000.
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