182 Bisphenol A, but not bisphenol S, affects key microRNAs during bovine oocyte maturation

Oocyte maturation involves crucial hormone-dependent events that are uniquely susceptible to toxic insults by endocrine disrupting chemicals (EDCs). Emerging evidence suggests that small RNAs, including microRNAs (miRNAs), may be key participants in the response to EDCs. Bisphenol A (BPA) and bisphenol S (BPS) are chemicals with detrimental health effects, with BPA negatively affecting oocyte quality. The mode of action of bisphenols at the epigenetic level is not clear. Several miRNAs have been identified as crucial regulators of gene expression during development. This study aimed to examine key miRNAs in response to BPA or BPS treatment during oocyte maturation. Primary forms (pri-miRNA) and mature forms of miR-21, miR-155, miR-34c, and miR-146a were quantified by quantitative (q)PCR in IVM bovine cumulus-oocyte complexes (COCs) and invitro cultured (IVC) cumulus cells treated with BPA and BPS at physiologically significant doses (0.05mgmL−1). Total RNA, containing both forms of microRNAs, was isolated from pools of 40 COCs on a minimum of three biological replicates. Primary miRNAs and mature miRNAs were reverse transcribed (RT) using qScript cDNA and microRNA/cDNA kits, respectively, and quantified by qPCR, with three technical replicates for each biological one. In addition, mRNA and protein quantification of the downstream target DNMT3A in IVC cumulus cells further enhanced our understanding of EDC interference in epigenetic regulations in female reproduction. Total RNA was isolated from cumulus cells, mRNA (1μg) and mature miRNAs (0.5μg) were RT separately and cDNA was quantified by qPCR, as described above. Expression values were normalized against two housekeeping genes selected by GeNorm analysis. Twenty micrograms of proteins extracted by sonication were loaded on a 8% acrylamide gel and analysed by western blotting. Densitometry analysis was performed on 3 separate blots with protein levels normalized to the loading control, β-actin. One-way ANOVA was used to determine statistical differences among treatment groups with P<0.05 considered statistically significant. Results showed that BPA significantly increased miR-21 in COCs (P=0.02) and cumulus cells (P=0.01), increased pri-miR-21 in oocytes (P=0.03), suppressed miR-34c in cumulus cells (P=0.02), increased miR-155 in denuded oocytes (P=0.04), and had no effect on miR-146a. No changes were observed in response to BPS. Experiments in IVC cumulus cells showed similar miRNA profiles: miR-21 and miR-155 were significantly overexpressed in BPA-treated cells (P=0.04); however, miR-34c and miR-146a were not affected. Messenger RNA levels of DNMT3A increased (P=0.02) and protein levels of DNMT3A decreased in response to BPA (P=0.005). Overall, this study presents novel findings of BPA-induced miRNA dysregulation in IVM bovine oocytes and in IVC bovine cumulus cells. We can speculate that miR-21 participates in the BPA-induced dysregulation of DNMT3A, contributing to decreased fertility. This study did not show any effect of BPS on microRNA expression, suggesting an alternative mechanistic pathway for this analogue.

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Hypoxia-inducible factor (HIF1alpha) inhibition modulates cumulus cell function and affects bovine oocyte maturation in vitro†

Biology of Reproduction ◽

10.1093/biolre/ioaa196 ◽

2020 ◽

Author(s):

Aslihan Turhan ◽

Miguel Tavares Pereira ◽

Gerhard Schuler ◽

Ulrich Bleul ◽

Mariusz P Kowalewski

Keyword(s):

Oocyte Maturation ◽

Cell Function ◽

Hypoxia Inducible Factor ◽

Cumulus Cell ◽

Cumulus Cells ◽

Negative Effects ◽

Protein Levels ◽

Maturation In Vitro

Abstract Various metabolic and hormonal factors expressed in cumulus cells are positively correlated with the in vitro maturation (IVM) of oocytes. However, the role of hypoxia sensing both during maturation of cumulus–oocyte complexes (COCs) as well as during the resumption of meiosis remains uncertain. HIF1alpha plays major roles in cellular responses to hypoxia, and here we investigated its role during bovine COC maturation by assessing the expression of related genes in cumulus cells. COCs were divided into the following groups: immature (control), in vitro matured (IVM/control), or matured in the presence of a blocker of HIF1alpha activity (echinomycin, IVM/E). We found an inhibition of cumulus cell expansion in IVM/E, compared with the IVM/control. Transcript levels of several factors (n = 13) were assessed in cumulus cells. Decreased expression of HAS2, TNFAIP6, TMSB4, TMSB10, GATM, GLUT1, CX43, COX2, PTGES, and STAR was found in IVM/E (P < 0.05). Additionally, decreased protein levels were detected for STAR, HAS2, and PCNA (P < 0.05), while activated-Caspase 3 remained unaffected in IVM/E. Progesterone output decreased in IVM/E. The application of PX-478, another blocker of HIF1alpha expression, yielded identical results. Negative effects of HIF1alpha suppression were further observed in the significantly decreased oocyte maturation and blastocyst rates from COCs matured with echinomycin (P < 0.05) or PX-478 (P < 0.05). These results support the importance of HIF1alpha for COC maturation and subsequent embryo development. HIF1alpha is a multidirectional factor controlling intercellular communication within COCs, steroidogenic activity, and oocyte development rates, and exerting effects on blastocyst rates.

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249 THE DIRECT AND CUMULUS-MEDIATED EFFECTS OF PROLACTIN ON BOVINE OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv21n1ab249 ◽

2009 ◽

Vol 21 (1) ◽

pp. 222 ◽

Cited By ~ 1

Author(s):

I. Lebedeva ◽

M. Vejlsted ◽

N. Volkova ◽

G. Singina ◽

M. Schmidt

Keyword(s):

Messenger Rna ◽

Technical Assistance ◽

Calf Serum ◽

Cumulus Cells ◽

Pcr Analysis ◽

Female Reproduction ◽

System 2 ◽

System 1 ◽

Bovine Oocytes ◽

Serum System

Prolactin (PRL) is an important regulator of female reproduction, as indicated using PRL receptor knockout mice (Bole-Feysot C et al. 1998 Endocrinol. Rev. 19, 225–268). The presence of PRL receptors or their mRNA has been shown in different follicular compartments of various mammalian species, including oocytes of sheep and mice (Picazo RA et al. 2004 Reproduction 128, 545–553; Kiapekou E et al. 2005 Reprod. Biomed. Online 10, 339–346). The aim of the present study was to characterize the direct and cumulus-mediated pathways of PRL signaling into bovine oocytes. The expression of PRL receptor mRNA in follicular cells was detected by reverse transcription polymerase chain reaction (RT-PCR) analysis. For oocytes, nested PCR was used. In addition, a total of 707 COC and 689 denuded oocytes (DO) from follicles 2 to 8 mm in diameter were cultured for 6 h (COC), 8 h (DO), or 24 h (COC and DO) in the presence or absence of 50 ng mL–1 bovine PRL (20 IU mg–1; Research Center for Endocrinology, Moscow, Russia). The following systems for COC and DO culture were used: (1) TCM-199 containing 10% fetal calf serum (system 1) and (2) DMEM containing 10 U mL–1 pregnant mare’s serum gonadotropin (PMSG), 5 U mL–1 hCG (Intervet Scandinavia, Copenhagen, Denmark), and 5% estrous cow serum (system 2). The nuclear status of oocytes was evaluated by Tarkowski’s cytogenetic method. After IVM in system 2, a portion of the oocytes (348 COC and 311 DO) underwent IVF and IVC, and the embryo development was tracked until the blastocyst stage. All treatments were repeated 4 to 7 times. Results were expressed as mean ± SEM. Arcsine-transformed data were analyzed by two-way ANOVA. Messenger RNA expression of long and short isoforms of PRL receptor was revealed in both bovine oocytes and cumulus cells. In system 1, PRL raised the rate of DO remaining at the diplotene stage by 8 h of culture (from 32.6 ± 3.0 to 47.5 ± 4.7%, P < 0.05), whereas there was no effect of PRL on the meiotic resumption in COC. By contrast, the hormone exerted a stimulatory action on the meiotic progression in the presence of cumulus cells by increasing the proportion of oocytes reaching the telophase I or metaphase II stages during 24 h of maturation (from 61.9 ± 1.2 to 73.5 ± 0.9%, P < 0.05). In system 2, PRL did not affect nuclear maturation of either cumulus-enclosed or cumulus-free oocytes, with the maturation rate varying between 93.9 ± 2.7 and 95.2 ± 2.4% (COC) and between 85.6 ± 3.0 and 83.0 ± 4.0% (DO). When added to IVM system 2, PRL raised the cleavage and blastocyst rates only in the case of COC (from 72.2 ± 2.4 to 80.2 ± 2.1%, P < 0.05, and from 20.5 ± 3.6 to 40.9 ± 4.2%, P < 0.01, respectively). These findings suggest functioning of the direct and cumulus-mediated pathways of PRL signaling into bovine oocytes, with the hormone affecting meiosis via both pathways. We thank A. D. Roed for expert technical assistance with IVF and IVC. This research was supported in part by RFBR (project no. 07-04-01485). I.L. was the recipient of an Erasmus Mundus fellowship.

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176 REGULATORY ROLE OF miR-20a DURING BOVINE OOCYTE MATURATION

Reproduction Fertility and Development ◽

10.1071/rdv29n1ab176 ◽

2017 ◽

Vol 29 (1) ◽

pp. 196 ◽

Cited By ~ 1

Author(s):

E. Andreas ◽

D. Salilew-Wondim ◽

F. Rings ◽

E. Held ◽

M. Hoelker ◽

...

Keyword(s):

Oocyte Maturation ◽

Expression Analysis ◽

Germinal Vesicle ◽

Cumulus Cells ◽

Fine Tuning ◽

Bovine Oocyte ◽

Total Rna ◽

Progesterone Synthesis ◽

Spent Media

The role of microRNA in oocyte maturation is mostly associated with optimal turnover of the accumulated maternal transcripts during their growth to allow maturation. MiR-20a is a member of the miR-17–92 cluster, which has been found to be differentially expressed in bovine granulosa cells derived from preovulatory dominant and subordinate follicles. Our recent study showed that miR-20a is involved in the regulation of granulosa cell proliferation, differentiation, and progesterone synthesis by targeting PTEN and BMPR2 genes. Here, we aimed to investigate the role of miR-20a in the bovine oocyte maturation processes. For this, cumulus-oocyte complexes (COC) were aspirated from small antral follicles (2–8 mm in diameter) and cultured in groups of 50 in 400 µL of maturation media (TCM-199 media supplemented with 12% oestrus cow serum and 10 µg/ml Follitropin®) at 39°C in a humidified atmosphere with 5% (vol/vol) CO2 in the air for 22 h. The cumulus cells and oocytes before (germinal vesicle) and after maturation (metaphase II) were mechanically separated in 0.1% hyaluronidase (in TCM-199 media). To study whether the presence of cumulus cells or oocyte has an impact on the miR-20a expression, we cultured oocytectomized cumulus cells and oocytes with and without their companion cells. Moreover, COC were co-cultured with miR-20a mimic, inhibitor, or corresponding controls to investigate the role of this miRNA in oocyte maturation. The total RNA from cumulus cells and oocytes was extracted using miRNeasy® mini kit (Qiagen GmbH, Hilden, Germany). Total RNA from respective samples was reverse transcribed for mRNA and microRNA expression analysis. Quantitative expression analysis was performed using StepOnePlus™ System (Applied Biosystems, Foster City, CA, USA) and subsequent data were analysed using a comparative cycle threshold method. The progesterone released in the spent media was measured using progesterone enzyme-linked immunosorbent assay kit (ENZO Life Sciences GmbH, Loerrach, Germany). Here, we found that miR-20a expression in cumulus cells increased (P < 0.05) during oocyte maturation. Conversely, miR-20a expression in metaphase II stage oocytes was significantly lower (P < 0.001) compared with the germinal vesicle stage. The absence of oocyte cytoplasm resulted in reduced miR-20a expression in cumulus cells. On the other hand, the absent of cumulus cells increased miR-20a expression in oocytes. The miR-20a expression revealed that the microRNA transduction is restricted in the cumulus cells. The overexpression of miR-20a increased oocyte maturation rate (P < 0.05) by 4.8% (as determined by extrusion of the polar body) and the expression of oocyte maturation-related genes (INHBA, MAPK1, PTGS2, PTX3, and EGFR). The progesterone released in spent media of COC co-cultured with miR-20a mimic and inhibitor showed increasing (P = 0.0936) and decreasing (P = 0.0993) trends, respectively. In this study, we also found that miR-20a modulation altered the expression of PTEN and BMPR2 in cumulus cells. In conclusion, the modulation of miR-20a expression in cumulus cells regulates the oocyte maturation and partially involved in the progesterone synthesis by fine-tuning the expression of PTEN and BMPR2 genes.

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159 Effect of bisphenol A and bisphenol S on AMH and AMHR mRNA expression during in vitro bovine oocyte maturation and early embryo development

Reproduction Fertility and Development ◽

10.1071/rdv31n1ab159 ◽

2019 ◽

Vol 31 (1) ◽

pp. 204 ◽

Cited By ~ 1

Author(s):

A. Saleh ◽

L. Favetta

Keyword(s):

Bisphenol A ◽

Mrna Expression ◽

Cumulus Cells ◽

Early Embryo ◽

Receptor Expression ◽

Mrna Levels ◽

Bisphenol S ◽

Tissue Samples ◽

Receptor Mrna

Exposure to chemicals with known endocrine-disrupting effects, such as bisphenol A (BPA) and bisphenol S (BPS), leads to repercussions on oocyte development and, ultimately, on fertility. Bisphenol A is a plasticizer used worldwide that has been detected in blood, urine, tissue samples and follicular fluid. Due to its widely reported detrimental effects, BPA has been substituted with its analogue BPS. Previous experiments in our laboratory have shown that exposure of bovine oocytes to physiologically relevant doses of BPA resulted in spindle abnormalities, reduced meiosis progression, decreased blastocyst rate and gene expression changes. However, the effects of BPS have not yet been investigated. Anti-Müllerian hormone (AMH) has been reported to be a good marker of ovarian reserve and oocyte developmental capability and is commonly used in assisted reproduction for diagnostic measurements. There is evidence that women undergoing IVF with higher BPA levels have lower AMH levels and pregnancy success. The aim of this study was to assess the effect of BPA and BPS on AMH and its receptor as measures of oocyte developmental capability. Abattoir-derived bovine cumulus-oocyte complexes (COC) were matured in vitro in 4 groups: (1) control, (2) vehicle (0.1% ethanol), (3) BPA (0.05mg mL−1 in 0.1% ethanol), and (4) BPS (0.05mg mL−1 in 0.1% ethanol). Pools of 30 COC, 30 denuded oocytes, and cumulus cells corresponding to denuded oocytes were collected for each of the 4 experimental groups, and a minimum of 4 biological replicates were used for each analysis. Anti-Müllerian hormone and AMH receptor mRNA expression was measured in COC, denuded oocytes, and their corresponding cumulus cells using quantitative real-time PCR. Statistical analyses were performed using 1-way ANOVA. Results showed a decrease (P<0.05) in AMH mRNA expression in BPA-treated oocytes (without cumulus cells). In addition, there was an increase (P<0.05) in mRNA AMH receptor levels in COC when treated with BPS. Finally, analyses on cumulus cells alone showed an increase (P<0.05) in the AMH receptor mRNA levels in BPA-treated cells. These results suggest that BPA has an effect on AMH mRNA transcript levels in oocytes, while affecting the receptor expression in cumulus cells. Conversely, BPS affects AMH indirectly, increasing the mRNA levels of its receptor only. Further investigation of the effects of BPA and BPS on AMH expression in in vitro-produced blastocysts derived from treated oocytes will aid in understanding the potential consequences of exposure to BPA and its analogues on early embryonic development.

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Oocyte-Secreted Factors Strongly Stimulate sFRP4 Expression in Human Cumulus Cells

MHR Basic science of reproductive medicine ◽

10.1093/molehr/gaab031 ◽

2021 ◽

Author(s):

Sahar Esfandyari ◽

Nicola J Winston ◽

Michelle A Fierro ◽

Humberto Scoccia ◽

Carlos Stocco

Keyword(s):

Regulatory Protein ◽

Cumulus Cells ◽

Mrna Levels ◽

Dependent Manner ◽

Female Reproduction ◽

Concentration Dependent Manner ◽

Protein Levels ◽

Fertility Treatments ◽

Secreted Factors ◽

Steroidogenic Acute Regulatory

Abstract Secreted frizzled-related protein-4 (SFRP4) belongs to a family of soluble ovarian-expressed proteins that participate in female reproduction, particularly in rodents. In humans, SFRP4 is highly expressed in cumulus cells. However, the mechanisms that stimulate SFRP4 in cumulus cells have not been examined. We hypothesise that oocyte-secreted factors such as growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are involved in the regulation of SFRP4. Human cumulus cells were collected from patients undergoing fertility treatments and treated with GDF9 or BMP15 or their combination in the presence of follicle-stimulating hormone (FSH) or vehicle. FSH treatment significantly decreased SFRP4 mRNA levels when compared with nontreated cells. However, SFRP4 mRNA levels were increased significantly by GDF9 plus BMP15 in a concentration-dependent manner in the presence or absence of FSH. The combination of GDF9 plus BMP15 also increased SFRP4 protein levels and decreased the activity of the β-catenin/TCF-responsive promoter significantly. GDF9 plus BMP15 inhibited steroidogenic acute regulatory protein and luteinising hormone/choriogonadotrophin (LH/hCG) receptor stimulation by FSH, while treatment with SFRP4 blocked the stimulatory effect of FSH on these genes. The evidence demonstrates that GDF9 and BMP15 act in coordination to stimulate SFRP4 expression and suggests that SFRP4 mediates the anti-luteinising effects of the oocyte in human cumulus cells.

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Expression and Impact of Vaspin on In Vitro Oocyte Maturation through MAP3/1 and PRKAA1 Signalling Pathways

International Journal of Molecular Sciences ◽

10.3390/ijms21249342 ◽

2020 ◽

Vol 21 (24) ◽

pp. 9342

Author(s):

Patrycja Kurowska ◽

Ewa Mlyczyńska ◽

Anthony Estienne ◽

Alix Barbe ◽

Iwona Rajska ◽

...

Keyword(s):

Protein Expression ◽

Oocyte Maturation ◽

Control Level ◽

Cumulus Cells ◽

In Vitro Fertilisation ◽

Female Reproduction ◽

Dapi Staining ◽

Compound C ◽

In Vitro Oocyte Maturation

Oocyte maturation is a critical stage in embryo production and female reproduction. The aims of this study were to determine: (i) the mRNA and protein expression of vaspin and its receptor 78-kDa glucose-regulated (GRP78) in porcine cumulus–oocyte complexes (COCs) by real-time PCR and Western blot analysis, respectively, and their localisation by immunofluorescence; and (ii) the effects of vaspin on in vitro oocyte maturation (IVM) and the involvement of mitogen ERK1/2 (MAP3/1)- and AMPKα (PRKAA1)-activated kinases in the studied processes. Porcine COCs were matured in vitro for 22 h or 44 h with vaspin at a dose of 1 ng/mL and nuclear maturation assessed by Hoechst 33342 or DAPI staining and the measurement of progesterone (P4) level in the maturation medium. We showed that vaspin and GRP78 protein expression increased in oocytes and cumulus cells after IVM. Moreover, vaspin enhanced significantly porcine oocyte IVM and P4 concentration, as well as MAP3/1 phosphorylation, while decreasing PRKAA1. Using pharmacological inhibitors of MAP3/1 (PD98059) and PRKAA1 (Compound C), we observed that the effect of vaspin was reversed to the control level by all studied parameters. In conclusion, vaspin, by improving in vitro oocyte maturation via MAP3/1 and PRKAA1 kinase pathways, can be a new factor to improve in vitro fertilisation protocols.

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Effects of BPA, BPS, and BPF on Oxidative Stress and Antioxidant Enzyme Expression in Bovine Oocytes and Spermatozoa

Genes ◽

10.3390/genes13010142 ◽

2022 ◽

Vol 13 (1) ◽

pp. 142

Author(s):

Mimi Nguyen ◽

Reem Sabry ◽

Ola S. Davis ◽

Laura A. Favetta

Keyword(s):

Oxidative Stress ◽

Bisphenol A ◽

Western Blot ◽

Enzyme Expression ◽

Bisphenol S ◽

Protein Levels ◽

Bisphenol F ◽

Mrna And Protein Expression ◽

Bovine Oocytes ◽

Ros Scavengers

Bisphenol A (BPA) and its analogs, bisphenol S (BPS) and bisphenol F (BPF), might impact fertility by altering oxidative stress pathways. Here, we hypothesize that bisphenols-induced oxidative stress is responsible for decreased gamete quality. In both female (cumulus-oocyte-complexes—COCs) and male (spermatozoa), oxidative stress was measured by CM-H2DCFDA assay and key ROS scavengers (SOD1, SOD2, GPX1, GPX4, CAT) were quantified at the mRNA and protein levels using qPCR and Western blot (COCs)/immunofluorescence (sperm). Either gamete was treated in five groups: control, vehicle, and 0.05 mg/mL of BPA, BPS, or BPF. Our results show elevated ROS in BPA-treated COCs but decreased production in BPS- and BPF-treated spermatozoa. Additionally, both mRNA and protein expression of SOD2, GPX1, and GPX4 were decreased in BPA-treated COCs (p < 0.05). In sperm, motility (p < 0.03), but not morphology, was significantly altered by bisphenols. SOD1 mRNA expression was significantly increased, while GPX4 was significantly reduced. These results support BPA’s ability to alter oxidative stress in oocytes and, to a lesser extent, in sperm. However, BPS and BPF likely act through different mechanisms.

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Bisphenol A and S impaired ovine granulosa cell steroidogenesis

Reproduction ◽

10.1530/rep-19-0575 ◽

2020 ◽

Vol 159 (5) ◽

pp. 571-583 ◽

Cited By ~ 1

Author(s):

Ophélie Téteau ◽

Manon Jaubert ◽

Alice Desmarchais ◽

Pascal Papillier ◽

Aurélien Binet ◽

...

Keyword(s):

Gene Expression ◽

Bisphenol A ◽

Food Industry ◽

Female Reproduction ◽

Bisphenol S ◽

Steroidogenic Enzyme ◽

Structural Analogue ◽

Steroid Production ◽

Safe Alternative ◽

Progesterone Secretion

Bisphenols, plasticisers used in food containers, can transfer to food. Bisphenol A (BPA) has been described as an endocrine disruptor and consequently banned from the food industry in several countries. It was replaced by a structural analogue, Bisphenol S (BPS). BPA action on the steroidogenesis is one of the mechanisms underlying its adverse effects on the efficiency of female reproduction. This study aimed to determine whether BPS is a safe alternative to BPA regarding GC functions. Antral follicles (2–6 mm), of approximatively 1000 adult ewe ovaries, were aspired and GC purified. For 48 h, ovine GC were treated with BPA or BPS (from 1 nM to 200 µM) and the effects on cell viability, proliferation, steroid production, steroidogenic enzyme expression and signalling pathways were investigated. Dosages at and greater than 100 μM BPA and 10 µM BPS decreased progesterone secretion by 39% (P < 0.001) and 22% (P = 0.040), respectively. BPA and BPS 10 μM and previously mentioned concentrations increased oestradiol secretion two-fold (P < 0.001 and P = 0.082, respectively). Only 100 µM BPA induced a decrease (P < 0.001) in gene expression of the enzymes of steroidogenesis involved in the production of progesterone. BPA reduced MAPK3/1 phosphorylation and ESR1 and ESR2 gene expression, effects that were not observed with BPS. BPA and BPS altered steroidogenesis of ovine GC. Thus, BPS does not appear to be a safe alternative for BPA. Further investigations are required to elucidate BPA and BPS mechanisms of action.

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