- Open Access
Cellular and molecular evidence for a role of tumor necrosis factor alpha in the ovulatory mechanism of trout
© Crespo et al; licensee BioMed Central Ltd. 2010
Received: 15 February 2010
Accepted: 12 April 2010
Published: 12 April 2010
The relevance of immune-endocrine interactions to the regulation of ovarian function in teleosts is virtually unexplored. As part of the innate immune response during infection, a number of cytokines such as tumor necrosis factor alpha (TNF alpha) and other immune factors, are produced and act on the reproductive system. However, TNF alpha is also an important physiological player in the ovulatory process in mammals. In the present study, we have examined for the first time the effects of TNF alpha in vitro in preovulatory ovarian follicles of a teleost fish, the brown trout (Salmo trutta).
To determine the in vivo regulation of TNF alpha expression in the ovary, preovulatory brook trout (Salvelinus fontinalis) were injected intraperitoneally with either saline or bacterial lipopolysaccharide (LPS). In control and recombinant trout TNF alpha (rtTNF alpha)-treated brown trout granulosa cells, we examined the percentage of apoptosis by flow cytometry analysis and cell viability by propidium iodide (PI) staining. Furthermore, we determined the in vitro effects of rtTNF alpha on follicle contraction and testosterone production in preovulatory brown trout ovarian follicles. In addition, we analyzed the gene expression profiles of control and rtTNF alpha-treated ovarian tissue by microarray and real-time PCR (qPCR) analyses.
LPS administration in vivo causes a significant induction of the ovarian expression of TNF alpha. Treatment with rtTNF alpha induces granulosa cell apoptosis, decreases granulosa cell viability and stimulates the expression of genes known to be involved in the normal ovulatory process in trout. In addition, rtTNF alpha causes a significant increase in follicle contraction and testosterone production. Also, using a salmonid-specific microarray platform (SFA2.0 immunochip) we observed that rtTNF alpha induces the expression of genes known to be involved in inflammation, proteolysis and tissue remodeling. Furthermore, the expression of kallikrein, TOP-2, serine protease 23 and ADAM 22, genes that have been postulated to be involved in proteolytic and tissue remodeling processes during ovulation in trout, increases in follicles incubated in the presence of rtTNF alpha.
In view of these results, we propose that TNF alpha could have an important role in the biomechanics of follicle weakening, ovarian rupture and oocyte expulsion during ovulation in trout, primarily through its stimulation of follicular cell apoptosis and the expression of genes involved in follicle wall proteolysis and contraction.
Fish are exposed to a variety of pathogens and stressful conditions which may cause severe reproductive consequences [1, 2]. It is now known that activation of the immune system as a result of a bacterial infection is characterized by the production of a wide variety of immune factors, including pro-inflammatory cytokines and chemokines  which can act as local chemical modulators or be secreted into the circulation and act as hormones. In particular, exposure to lipopolysaccharide (LPS), an active component of the wall of Gram-negative bacteria, induces the expression of TNFα in trout macrophages . Once secreted into the circulation, the pleiotropic cytokine TNFα can then act on a number of different tissues, including non-immune tissues.
In mammals, TNFα is known to act in the ovary, where it affects differentiation, proliferation, steroid production, inflammation and induction of apoptosis through specific receptors [5–7]. Interestingly, TNFα is considered to be a mediator of ovulation through its stimulatory role on follicular apoptosis and breakdown of the extracellular matrix (ECM) in the follicle wall [8, 9]. Therefore, in addition to its local production and action, TNFα can act as a mediator signaling the status of the immune system to the reproductive system. In fish, very little is known about the interaction between the immune and the reproductive systems and, in particular, of the involvement of TNFα in the reproductive process. We have previously reported that LPS administration results in the induction of apoptosis in the trout ovary and that preovulatory trout follicles incubated with conditioned medium from LPS-stimulated trout macrophages showed an increase in follicle contraction, suggesting that factors produced by trout macrophages in response to LPS may stimulate follicle contraction . In view of these observations, we hypothesize that TNFα could be one of the most likely mediators of the effects of LPS in the trout ovary. Therefore, it is important to address the issue of the impact of the activation of the immune response by TNFα on reproductive function in fish.
In the present study, we have examined the effects (in vitro) of recombinant trout TNFα (rtTNFα) on the reproductive function of preovulatory brown trout (Salmo trutta) at various levels: (1) ovarian apoptosis and granulosa cell viability, (2) follicle contraction and steroid production and (3) gene expression profiles (microarray and qPCR analyses).
Reproductively mature female brown trout (Salmo trutta) from a cultured stock at the Piscifactoria de Bagà (Generalitat de Catalunya, Bagà, Spain) were kept under natural conditions of temperature and photoperiod. Fish at the preovulatory stage (according to the position of the germinal vesicle (GV) in oocytes that were cleared using a solution previously described ), were anesthesized in 3-aminobenzoic acid ethyl ester (0.1 g/l; Sigma, Alcobendas, Spain) dissolved in fresh water, and the fish were sacrificed by concussion prior to the collection of the ovaries. The dissected ovaries were immediately used for the various in vitro assays. For the experiment on the in vivo regulation of TNFα expression in the ovary, preovulatory brook trout (Salvelinus fontinalis) were briefly anesthesized in 3-aminobenzoic acid ethyl ester (0.1 g/l) and injected intraperitoneally with either saline (n = 3) or E. coli lipopolysaccharide (LPS) (3 mg/kg weight; Sigma) (n = 3) once a day over four consecutive days, as previously reported . Twenty four hours after the last injection, fish were anesthesized (as described above) and sacrificed by spinal transection prior to the collection of the ovaries. Ovarian tissue (previously de-yolked by gentle pressure) was flash frozen in liquid nitrogen and stored at -80°C, until RNA isolation.
Hormones and reagents
Coho salmon (Oncorhynchus kisutch) luteinizing hormone (sLH) was a kind gift from Dr. Penny Swanson (National Marine Fisheries Service, Seattle, WA)  and was dissolved directly in incubation medium.
For the generation of rtTNFα, the mature form of TNFα from brook trout (Salvelinus fontinalis) was subcloned into an expression vector, produced in E. coli, refolded, concentrated and shown to be biologically active using a fish cell line .
Ovarian tissue incubations
After dissection, brown trout preovulatory ovaries were placed in Hank's balanced salt solution (HBSS) and individual ovarian follicles were manually separated with forceps from each ovary on ice, as previously described . For the follicle contraction experiments, brown trout preovulatory follicles punctured using a 25-gauge hypodermic needle were incubated in HBSS containing 0.2% BSA (fraction V, Sigma; HBSS-BSA) in 6 cm culture dishes (10 follicles/4 ml) in the presence of the test compounds for 16 h at 15°C under shaking conditions (100 rpm). Follicle contraction was determined by measuring the weight of the 10 follicles in each replicate after incubation, as previously described and validated for epinephrine stimulation [15, 16]. Since contraction results in the expulsion of yolk through the puncture site, a decrease in follicle weight indicates an increase in follicle contraction. For in vitro steroid production experiments, intact preovulatory brown trout follicles were incubated (10 follicles/well/1 ml) in HBSS-BSA in 24-well culture plates, in the absence or presence of different test compounds for 24 h at 15°C with gentle shaking (100 rpm). At the termination of the incubation, the medium was removed and stored at -20°C, until assayed.
To collect ovarian tissue for RNA extraction, preovulatory follicles from each of a total of three females were incubated (400 follicles/50 ml) in HBSS-BSA in the absence or presence of rtTNFα (100 ng/ml, dissolved directly in HBSS-BSA), at 15°C for 24 h with gentle shaking (100 rpm). At the end of the incubation follicles (previously de-yolked by gentle pressure) were removed, flash frozen in liquid nitrogen and stored at -80°C until assayed.
The concentration of testosterone (T) in brown trout ovarian follicle incubates were measured directly using commercial radioimmunoassay (Schering-CIS, Madrid, Spain), as described previously [17, 18].
Analysis of apoptosis and cell viability
Granulosa cell viability was assessed by incubating brown trout ovarian follicles in the absence or presence of 100 ng/ml rtTNFα for 18 h at 15°C, separating the granulosa layers and staining them with propidium iodide (PI; 50 μg/ml; Sigma). Subsequently, sheets of granulosa layers were mounted onto glass slides, counterstained with DAPI and visualized under a fluorescent microscope. To determine the incidence of apoptosis after rtTNFα treatment, granulosa cells were obtained from granulosa layers (isolated from control and rtTNFα-treated follicles) by enzymatic digestion with 0.2% collagenase type IA (Clostridium histolyticum; Sigma) under gentle agitation for 2 h at room temperature. After incubation, the homogenate was filtered through a 100 μm filter and subsequently through a 40 μm filter before centrifugation for 6 min at 500 g. The pellet was washed with 0.5 ml 1 × PBS and centrifuged 5 min at 500 g. Isolated granulosa cells were resuspended in 70% ethanol and stored at -20°C until use. To analyze the incidence of apoptosis, granulosa cells were resuspended in PI staining buffer (20 μg/ml PI, 0.1% Triton X-100, 200 μg/ml RNase A; Sigma) prior to analysis by flow cytometry (FACS analysis).
RNA isolation and cDNA synthesis
Total RNA from ovarian tissue was isolated using TRIzol Reagent (Invitrogen, Barcelona, Spain), following the manufacturer's instructions and guidelines. Quantification was carried out with Qubit fluorometer (Invitrogen). cDNA synthesis was performed on 5 ìg DNase-treated (RQ1 DNase, Promega, Barcelona, Spain) total RNA using SuperScript III Transcriptase (Invitrogen), oligo(dT) primer and random hexamer primers (Promega), according to the manufacturer's protocols. The RNA was stored at -80°C until use.
Microarray analyses were performed using the salmonid fish cDNA microarray platform (SFA2.0 immunochip) previously validated and described [19, 20] that has been deposited in GEO under accession number GPL6154. Total RNA from pooled control and TNFα-treated brown trout preovulatory follicles (n = 20) from each of a total of three females was labelled with Cy3-dUTP and Cy5-dUTP (GE Healthcare, Barcelona, Spain) using SuperScript III Transcriptase. The cDNA synthesis reaction was performed at 50°C for 2 h in a 20 μl reaction volume, followed with RNA degradation with 0.2 M NaOH at 37°C for 15 min and alkaline neutralization with 0.6 M Hepes. Labeled cDNA was purified with Microcon YM30 (Millipore, Madrid, Spain). We used a dye swap experimental design and each pooled sample (20 follicles) from each female was hybridized to two microarrays. For the first slide, pooled test and control cDNA were labeled with Cy5 and Cy3 respectively, and for the second array dye assignment was reversed. The slides were pretreated with 1% BSA (fraction V), 5 × SSC, 0.1% SDS (30 min at 50°C) and washed with 2 × SSC (3 min) and 0.2 × SSC (3 min) at room temperature and hybridized overnight at 60°C in a cocktail containing 1.3 × Denhardt's, 3 × SSC, 0.3% SDS, 2.1 μg/μl polyadenylate and 1 μg/μl yeast tRNA. All chemicals were from Sigma. After hybridization, slides were washed at room temperature in 0.5 × SSC and 0.1% SDS for 15 min, 0.5 × SSC and 0.01% SDS for 15 min, and twice in 0.06 × SSC for 2 and 1 min, respectively. Scanning was performed with ScanArray 5000 (GSI Lumonics) and images were processed with GenePix Pro 5.0 (Axon). The measurements in spots were filtered by criteria I/B ≥ 3 and (I-B)/(S I + S B ) ≥ 0.6, where I and B are the mean signal and background intensities and S I , S B are the standard deviations. After subtraction of median background from median signal intensities, the expression ratios (ER) were calculated. Locally weighted non-linear regression (Lowess) normalization was performed, first for the whole slide and next for twelve rows and four columns per slide. The differential expression was assessed by the difference of the mean log2 ER from zero between the slides with reverse labelling (6 spot replicates per gene on each slide, Student's t-test, p < 0.01). The log2 ER ranked up- or down-regulated genes were analyzed interrogating the functional classes of Gene Ontology (GO)  and compared by the sums of ranked genes (Student's t-test, p < 0.05). Complete microarray results were submitted to NCBI GEO Omnibus (GSE20296).
Quantitative real-time PCR (qPCR)
Sequences of primers used in gene expression analyses by qPCR
Primer sequence (5'-3')
Amplicon size (bp)
Tumor necrosis factor alpha (TNFα)
Small inducible cytokine B14 precursor
Leukocyte cell-derived chemotaxin 2
TNF decoy receptor
Serine protease-like protein-1
Inhibitor of apoptosis protein 3
Novel osteopontin-like protein (NOP)
Trout ovulatory protein-2 (TOP-2)
Trout decoy receptor (TDcR)
Macrophage migration inhibitory factor (MIF)
18S ribosomal RNA
Differences among groups were analyzed for statistical significance with non parametric Mann-Whitney U test or by one-way analysis of variance (ANOVA) followed by the Fisher protected least significant different test, using StatView 5.0 (SAS Institute, Cary, NC). Results are expressed as means ± SE and differences between groups were considered to be significant if p < 0.05.
Effects of rtTNFα on ovarian apoptosis and viability
Effects of rtTNFα on follicle contraction
Effects of rtTNFα on steroid production
Effects of rtTNFα on ovarian gene expression
In order to evaluate the effects of rtTNFα treatment on gene expression in the trout ovary, we used a salmonid fish cDNA microarray platform (SFA2.0) enriched with immune-related genes that has been previously validated for studies involving response to stress and toxicity [19, 20] as well as the immune response in trout [10, 23–25].
Microarray analyses showed that several genes involved in apoptosis underwent changes in response to rtTNFα in vitro administration (Fig. 6), in support of the observed increase in apoptosis in rtTNFα-treated ovarian follicles (Fig. 2). Receptor-interacting serine/threonine-protein kinase 2 (cell death protein RIP) and growth arrest and DNA-damage inducible GADD45 beta, involved in TNFα-induced apoptosis, were significantly up-regulated. TNF-dependent genes increased their expression, including several receptors (TNF receptor superfamily members). Also, inhibitor of apoptosis protein 3 and cdk inhibitor p21 binding protein were significantly down-regulated after rtTNFα treatment. In addition, the expression of telomerase reverse transcriptase and nuclear matrix protein NMP200 were also reduced in the trout ovary. In mammals, loss of expression of these two genes results in apoptosis and decreased cell survival after DNA damage [26, 27]. Interestingly, genes involved in the cell cycle (such as cyclins) were reduced, in accordance with the GO categories regarding negative regulation of cell cycle and mitotic cell cycle.
Importantly, rtTNFα affected a number of genes involved in proteolysis, tissue remodeling and cytoskeleton (Fig. 6). This set of genes includes matrix metalloproteinase-2 (MMP-2 or gelatinase A), tissue inhibitor of metalloproteinase 2 (TIMP-2), plasminogen precursor-1, ADAMTS-3 (a disintegrin and metalloproteinase with thrombospondin-like motifs 3), serine proteases (serine protease-like protein-1 and 3), complement components (complement factor H-2, complement factor MASP-3, complement factor B/C2-B, complement component C9 and complement receptor 1-1), cathepsins (cathepsin D-2, C-2, B-2 and Y), cytoskeletal proteins and sarcomeric proteins involved in contractile function (actins, myosins and troponins).
Treatment with rtTNFα also caused an increase in the expression of immune-related genes in the trout ovary (Fig. 6 and Additional File 1, Figure S1). Several cytokines and chemokines, such as the small inducible cytokine B14 precursor (CXCL14), CCL4, leukocyte cell-derived chemotaxin 2 or chemokine-like factor family member 3 were induced. Furthermore, rtTNFα increased the expression of genes involved in communication and signaling, such as several MAP kinases and NF-kappaB inhibitor alpha transcripts (1 and 3). Also, genes coding for immune-related receptors showed changes in expression, such as chemokine 5a receptor-like, C type lectin receptor B and, as indicated above, several TNF receptor superfamily members.
qPCR validation of microarray results for selected genes
Small inducible cytokine B14 precursor
Leukocyte cell-derived chemotaxin 2
TNF decoy receptor
Serine protease-like protein-1
Inhibitor of apoptosis protein 3
In vitro effects of rtTNFα (100 ng/ml) on candidate gene expression in brown trout preovulatory follicles
Novel osteopontin-like protein (NOP)
Trout ovulatory protein-2 (TOP-2)
Trout decoy receptor (TDcR)
Macrophage migration inhibitory factor (MIF)
In mammals, it has been hypothesized that TNFα may be a component of the ovulatory mechanism [33, 34]. In fact, TNFα is produced in the ovary [35, 36] and its role in steroidogenesis, proliferation and follicular apoptosis has been studied in several species [37, 38]. Furthermore, ovulation is blocked after injection of TNFα antibodies into the follicular antrum . All these observations suggest that TNFα plays multiple physiological roles in ovarian function in mammals. In order to study the possible involvement of TNFα in ovarian function in fish, we have investigated for the first time the effects of rtTNFα on ovarian function in a teleost fish, the brown trout (Salmo trutta). In particular, we examined the in vitro effects of rtTNFα treatment on ovarian apoptosis and viability, follicle contraction, steroid production and gene expression.
One of the most significant results obtained in the present study is the stimulation of trout granulosa cell apoptosis by rtTNFα, as reported in mammals , and the modulatory effects of this cytokine on the expression of apoptosis-related genes in trout preovulatory follicles. Interestingly, the pro-apoptotic effects of rtTNFα at the cellular and molecular levels parallel those reported in the ovary of LPS-treated female trout . In fact, the expression of two anti-apoptotic genes (i.e. cdk inhibitor p21 binding protein and telomerase reverse transcriptase) in the preovulatory ovary was similarly down-regulated by in vivo administration of LPS and by in vitro treatment with rtTNFα. However, rtTNFα treatment also increased the expression of BCL-2 family members such as BCL2/adenovirus E1B 19-kDa protein-interacting protein 2 and BCL2-associated athanogene 1 (BAG1). BCL-2 interacting protein BAG-1 enhances the anti-apoptotic effects of BCL-2  and expression of BCL-2 in mouse oocytes during fetal development suppresses apoptotic cell death . Furthermore, the expression of ferritin heavy chain-1 was also increased by rtTNFα and its up-regulation is known to inhibit TNFα-induced apoptosis . These results could suggest the existence of a balance between multiple pro-apoptotic (such as cell death protein RIP, growth arrest and DNA-damage inducible GADD45 beta or nuclear protein 1) and anti-apoptotic genes regulated by TNFα around the time of ovulation that could be important in order to exert a tight, and perhaps localized, control of TNFα-induced apoptosis in the trout ovary.
In addition to the induction of apoptosis, treatment of trout preovulatory follicles with rtTNFα caused important changes in the expression of genes involved in proteolysis and remodeling of the ECM. In mammals, alterations in the ECM of the follicle wall are an essential part of ovulation. For the oocyte to be released, extensive breakdown of the collagenous tissue of the theca externa and of the granulosa cell basement membrane, as well as dissociation of granulosa cell layers, are required . In fish, there is also evidence for ECM remodeling during ovulation since it has been reported that MMPs with collagenase activity increase in the follicle wall at the time of ovulation in trout [43, 44]. Furthermore, up-regulation of other candidate genes possibly involved in ovarian proteolysis such as kallikrein (KT-14), a serine protease postulated to regulate oocyte expulsion in trout , TOP-2, a serine protease inhibitor [29, 45], as well as serine protease 23 and ADAM22, two genes identified by microarray analysis , occurs during natural ovulation in trout. In medaka (Oryzias latipes), several MMPs and TIMP-2 have been shown to play a crucial role in ECM remodeling, degradation of the follicle wall and, subsequently, ovulation, as in mammals . Specifically, MMP-2 (gelatinase A) and membrane-type 2 MMP facilitate follicle rupture during ovulation by degrading type IV and type I collagen, respectively, and TIMP-2 regulates the production and activity of MMP-2 in medaka preovulatory follicles . However, there is no evidence to date regarding the nature of the regulatory factors that control the production and activity of proteins involved in follicle rupture and ovulation in fish. In the present study we show that rtTNFα induces the expression of proteolytic enzymes in the trout ovary that belong to the three classes of proteinases known to be involved in ECM remodeling during ovulation in mammals: MMPs (e.g. MMP-2), plasmin/plasminogen activator system (e.g. plasminogen precursor-1) and ADAMTS (e.g. ADAMTS-3). In addition to these proteinases, rtTNFα induced the expression of TOP-2, TIMP-2, kallikrein and other serine proteases. Furthermore, we observed an increase in the expression of several cathepsins (D-2, C-2, B-2 and Y) in agreement with recent information suggesting an important role for cathepsins during ovarian follicle growth and maturation in fish . Therefore, we have evidence supporting the hypothesis that rtTNFα may be an important factor regulating the degradation of the follicle wall, as a prerequisite for oocyte release, in the trout ovary.
Here we also provide direct evidence for a stimulatory role of rtTNFα on follicle contraction, a process required for oocyte expulsion during ovulation. Increased follicle contraction is most likely related to the observed up-regulation of the expression of cytoskeletal and sarcomeric proteins involved in contractile function. In a previous study we suggested that the stimulation of trout follicle contraction by conditioned medium from LPS-stimulated trout macrophages was due to the presence of inflammatory cytokines in the medium . We have recently confirmed the ability of LPS-stimulated trout macrophages to secrete native TNFα protein  and, consequently, we can reasonably speculate that TNFα was at least one of the cytokines present in conditioned medium responsible for the stimulation of follicle contraction. In addition to TNFα, other factors such as chatecholamines and prostaglandins stimulate follicle contraction in trout [15, 16]. Future studies in our laboratory are devoted to investigate a possible link between TNFα and prostaglandins in the stimulation of follicle contraction in trout preovulatory follicles.
The stimulation of testosterone production by rtTNFα in trout preovulatory follicles is interesting in relation to the known potentiatory role of androgens in the ovulatory process in mammals . Although the precise mechanism by which androgens may induce ovulation in mammals is not well characterized, there is evidence for the increased expression of proteolytic enzymes such as kallikrein , suggesting that androgens could be involved in follicle rupture. In female salmonid fish (salmon and trout), testosterone plasma levels are known to increase significantly prior to ovulation [30, 50] but there is no functional evidence for a role of testosterone in ovulation in fish. Our results, supported by the fact that ovarian follicles from LPS-treated trout showed increased sLH-stimulated testosterone production , suggest that TNFα could be responsible, at least in part, for the high ovarian testosterone output in preovulatory trout. It is tempting to speculate that TNFα-induced testosterone could play a role in the ovulatory process in fish.
Based on our observations on the stimulatory effects of rtTNFα on trout granulosa cell apoptosis and on the expression of genes involved in proteolysis of the trout follicle wall, we suggest that, as described in mammals (reviewed in ), TNFα in trout could facilitate ovulation by serving the dual role of promoting collagen degradation in the follicle wall and further disrupting it by deleting follicular cells by apoptosis. This role of TNFα, coupled with its ability to directly stimulate follicle contraction in trout preovulatory follicles, strongly suggests that TNFα may have pro-ovulatory actions in fish. In support of the proposed role for TNFα in the ovulatory process in fish, inhibition of the activity of TNFα-converting enzyme (TACE/ADAM17), which inhibits TNFα secretion by trout macrophages , blocks ovulation in isolated medaka follicles . Furthermore, we have recently shown that LPS administration in vivo may result in an advancement of the time of ovulation in trout . Having demonstrated that LPS administration in vivo induces the expression of TNFα in the trout ovary (this study), we can speculate that ovarian production of TNFα may trigger the ovulatory process. At least in trout, the expression of ligands and receptors of the TNF family, most notably TNFα itself  and TDcR , which may serve to bind TNFα and control its activity, increase at the time of ovulation.
This is the first report in fish describing the possible role of TNFα in trout ovarian follicles during the preovulatory period. rtTNFα induced apoptosis in trout granulosa cells and altered the expression of genes known to be involved in the normal ovulatory process in trout, including genes involved in inflammation, proteolysis and tissue remodeling. Furthermore, rtTNFα stimulated the contraction of preovulatory trout follicles as well as the production of testosterone. These results suggest that TNFα, as in mammals, may be an important factor in the control of ovulation in fish by participating in the biomechanics of follicle weakening and rupture as well as in oocyte expulsion during ovulation. Furthermore, the results of the present study support the idea that bacterial pathogens, through the induction of pro-inflammatory cytokines such as TNFα, could have an impact on ovarian function, possibly resulting in an advancement of ovulation in fish, with important consequences for broodstock and egg quality in farmed fish.
We would like to thank Mr Antonino Clemente at the Piscifactoria de Bagà (Departament de Medi Natural, Generalitat de Catalunya, Spain) for providing the fish used in this study, and for his generous assistance. We would also like to thank the Turku Centre of Biotechnology for the preparation of microarrays and Dr Arjan Palstra for comments on the manuscript. This study was funded in part by grants from the Ministerio de Ciencia e Innovación (Acción Integrada HF2005-0030 and AGL2009-07006), Spain to JVP and from Egide, France to JB.
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