- Open Access
Cellular and molecular evidence for a role of tumor necrosis factor alpha in the ovulatory mechanism of trout
Reproductive Biology and Endocrinology volume 8, Article number: 34 (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)
In order to quantify mRNA expression of individual genes, quantitative real-time PCR (qPCR) was carried out with the same samples used for microarray analyses (pooled follicles from each of three separate females). cDNA was diluted 1:25 for target mRNA and 1:2000 for 18S and used as a template. The reactions (20 μl final volume) contained 10 μl of SYBR GreenER qPCR SuperMix (Invitrogen), 500 nM concentration of forward and reverse primers and 5 μl of cDNA. Reactions were run in a iCycler Thermal Cycler (BioRad, Alcobendas, Spain) under the following protocol: 2 min at 50°C, 8 min at 95°C, followed by 40 cycles of 15 sec denaturation at 95°C and 30 sec at 60°C, and a final melting curve of 81 cycles from 55°C to 95°C (0.5°C increments every 10 sec). All samples were run in triplicate and fluorescence was measured at the end of every extension step. Fluorescence readings were used to estimate the values for the threshold cycles (C t). Relative expression of mRNA was evaluated by -ΔΔC t, based on the quantification method by Livak and Schmittgen . As the reference gene, 18S ribosomal RNA was selected. For all primer pairs, a dilution curve obtained from a serially diluted ovarian cDNA pool was used to ensure that PCR efficiency was higher than 90%. Primer sequences, amplicon sizes and GenBank accession numbers of the target genes are presented in Table 1.
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.
In a previous study, we reported changes in ovarian function in trout that had been administered with LPS to simulate a bacterial infection . Specifically, we hypothesized that the induction of follicular apoptosis in LPS-injected females and the stimulation of follicle contraction by LPS-stimulated macrophage conditioned medium could have been caused by the increased local production of pro-apoptotic cytokines, such as TNFα. Therefore, in order to determine if TNFα could mediate the observed effects of LPS in the trout ovary, in the present study we investigated the direct biological effects of rtTNFα on trout preovulatory follicles. Importantly, we first demonstrate here that LPS administration in vivo causes a significant induction (1.99-fold; p < 0.05) of the ovarian expression of TNFα in brook trout (Fig. 1), the species used in our previous study (8). We have also confirmed the stimulation of ovarian TNFα expression by LPS administration in rainbow trout (results not shown), which suggest that this effect may not be species-specific.
Effects of rtTNFα on ovarian apoptosis and viability
We investigated whether rtTNFα can induce apoptosis and/or cause a decrease in the viability of trout granulosa cells. Our results clearly show that the incidence of apoptotic granulosa cells significantly (p < 0.05) increased in trout ovarian follicles incubated with rtTNFα (Fig. 2A). Evaluation of granulosa cell viability by PI staining provided additional evidence that exposure of trout ovarian follicles to rtTNFα in vitro causes a marked decrease in granulosa cell viability (Fig. 2B). In contrast to the untreated controls in which almost no PI-positive cells were detected (Fig. 2B), treatment with rtTNFα increased the presence of PI-positive cells (Fig. 2B). Exposure of granulosa cells to 1% paraformaldehyde (PFA), used as a positive control, also caused a decrease in cell viability (Fig. 2B).
Effects of rtTNFα on follicle contraction
Follicle contraction was significantly (p < 0.05) stimulated in isolated trout preovulatory follicles incubated in the presence of rtTNFα (Fig. 3), as evidenced by the decrease in follicle weight when compared with untreated unpunctured controls (UP controls) and punctured controls (P controls). Epinephrine (10 μM), used as a positive control, also significantly (p < 0.05) stimulated follicle contraction. There was approximately a 12% spontaneous contraction in punctured control follicles, as shown in previous studies [15, 16].
Effects of rtTNFα on steroid production
Incubation with rtTNFα stimulated the in vitro production of T in a concentration-dependent manner (significantly at 10 and 50 ng/ml; p < 0.05) (Fig. 4A) by intact trout follicles. As expected, sLH (25 ng/ml) significantly (p < 0.05) stimulated the production of T. In the same manner, rtTNFα caused a significant (p < 0.05) potentiation of the effects of sLH on T production (Fig. 4B).
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].
Overall, a larger number of genes were up-regulated (n = 235) rather than down-regulated (n = 158) in trout ovarian follicles in response to rtTNFα treatment (Fig. 5 and Additional File 1, Figure S1). However, applying a cut-off value of > 1 log2 ER (expression ratio) over the differentially expressed genes (DEGs) (p < 0.01) resulted in a considerably higher number of genes being down-regulated (n = 28) as opposed to those up-regulated (n = 6) (Fig. 5). Furthermore, the highest ERs in individual genes were observed among the down-regulated genes (Additional File 1, Figure S1). Fig. 6 shows examples of DEGs with known functions based on gene ontology.
The DEGs that were identified in response to rtTNFα treatment in the trout ovary were analyzed by interrogating the functional classes of Gene Ontology (GO)  and compared by the mean log2 ER of ranked genes (Student's t-test, p < 0.05) (Fig. 7 and Additional File 2, Figure S2A and B). The categories G1/S transition of mitotic cell cycle, nucleotide metabolism, DNA replication and mitotic cell cycle were reduced by rtTNFα, whereas protein biosynthesis and structural constituent of ribosome were enhanced. Also, categories related to protein kinase and I-kappaB kinase NF-kappaB cascades and apoptosis were up-regulated. Furthermore, categories involved in tissue remodeling, cytoskeleton and muscle contraction were also increased.
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.
In the present study, we selected eight differentially expressed genes representing varying levels of expression to verify the microarray results by qPCR (Table 2) using the same experimental treatments. Results obtained by qPCR validated the data obtained from microarray analysis. Genes such as small inducible cytokine B14 precursor, CCL4 and triosephosphate isomerase were highly regulated in the qPCR analysis.
Also, we identified by qPCR (Table 3) the expression of genes that have been postulated to be involved in proteolytic and tissue remodeling processes during ovulation in trout (i.e. kallikrein, KT-14; trout ovulatory protein-2, TOP-2; and novel osteopontin-like protein, NOP) [28–30]. The expression of this set of genes was significantly (p < 0.05) increased over control in response to rtTNFα treatment (100 ng/ml). Furthermore, we analyzed two interesting genes that are also expressed during the ovulatory process, such as macrophage migration inhibitory factor (MIF), a cytokine stimulated in response to human chorionic gonadotropin (hCG) or LH in follicular fluid , and TNFα trout decoy receptor (TDcR), a soluble receptor detected in the ovary during and after natural ovulation  (Table 3). The expression of both genes was significantly (p < 0.05) increased by rtTNFα.
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.
Currie JL, Woo PTK: Effects of the pathogenic haemoflagellate, Cryptobia salmositica on brood fish, Oncorhynchus mykiss. Environ Biol Fish. 2008, 83 (3): 355-365. 10.1007/s10641-008-9350-x.
Schreck CB, Contreras-Sanchez W, Fitzpatrick MS: Effects of stress on fish reproduction, gamete quality, and progeny. Aquaculture. 2001, 197 (1-4): 3-24. 10.1016/S0044-8486(01)00580-4.
Goetz FW, Iliev DB, McCauley LA, Liarte CQ, Tort LB, Planas JV, MacKenzie S: Analysis of genes isolated from lipopolysaccharide-stimulated rainbow trout (Oncorhynchus mykiss) macrophages. Mol Immunol. 2004, 41 (12): 1199-1210. 10.1016/j.molimm.2004.06.005.
MacKenzie S, Planas JV, Goetz FW: LPS-stimulated expression of a tumor necrosis factor-alpha mRNA in primary trout monocytes and in vitro differentiated macrophages. Dev Comp Immunol. 2003, 27 (5): 393-400. 10.1016/S0145-305X(02)00135-0.
Calleja-Agius J, Muttukrishna S, Jauniaux E: Role of TNF-α in human female reproduction. Expert Rev Endocrinol Metab. 2009, 4: 273-282. 10.1586/eem.09.4.
Amsterdam A, Keren-Tal I, Aharoni D, Dantes A, Land-Bracha A, Rimon E, Sasson R, Hirsh L: Steroidogenesis and apoptosis in the mammalian ovary. Steroids. 2003, 68 (10-13): 861-867. 10.1016/j.steroids.2003.09.003.
Roby KF, Terranova PF: Effects of tumor necrosis factor-alpha in vitro on steroidogenesis of healthy and atretic follicles of the rat: theca as a target. Endocrinology. 1990, 126 (5): 2711-2718. 10.1210/endo-126-5-2711.
Murdoch WJ, Colgin DC, Ellis JA: Role of tumor necrosis factor-alpha in the ovulatory mechanism of ewes. J Anim Science. 1997, 75 (6): 1601-1605.
Murdoch WJ, Lund SA: Prostaglandin-independent anovulatory mechanism of indomethacin action: inhibition of tumor necrosis factor alpha-induced sheep ovarian cell apoptosis. Biol Reprod. 1999, 61 (6): 1655-1659. 10.1095/biolreprod61.6.1655.
MacKenzie S, Montserrat N, Mas M, Acerete L, Tort L, Krasnov A, Goetz FW, Planas JV: Bacterial lipopolysaccharide induces apoptosis in the trout ovary. Reprod Biol Endocrinol. 2006, 4: 46-10.1186/1477-7827-4-46.
Goetz FW, Bergman HL: The effects of steroids on final maturation and ovulation of oocytes from brook trout (Salvelinus fontinalis) and yellow perch (Perca flavescens). Biol Reprod. 1978, 18 (2): 293-298. 10.1095/biolreprod18.2.293.
Swanson P, Suzuki K, Kawauchi H, Dickhoff WW: Isolation and characterization of two coho salmon gonadotropins, GTH I and GTH II. Biol Reprod. 1991, 44 (1): 29-38. 10.1095/biolreprod44.1.29.
Roher N, Callol A, Planas JV, Goetz FW, MacKenzie SA: Endotoxin recognition in fish results in inflammatory cytokine secretion not gene expression. Innate Immun. 2010, 10.1177/1753425909348232.
Planas JV, Goetz FW, Swanson P: Stimulation of brook trout ovarian steroidogenesis by gonadotropins I and II is mediated by the cyclic adenosine 3',5'-monophosphate/protein kinase A pathway. Biol Reprod. 1997, 57 (3): 647-654. 10.1095/biolreprod57.3.647.
Goetz FW, Bradley JA: Stimulation of in vitro ovulation and contraction of brook trout (Salvelinus fontinalis) follicles by adrenaline through alpha-adrenoreceptors. J Reprod Fertil. 1994, 100 (2): 381-385.
Hsu SY, Goetz FW: The effects of E and F prostaglandins on ovarian cAMP production and follicular contraction in the brook trout (Salvelinus fontinalis). Gen Comp Endocrinol. 1992, 88 (3): 434-443. 10.1016/0016-6480(92)90238-F.
Méndez E, Mæland M, Skålhegg BS, Planas JV: Activation of the cAMP-dependent protein kinase signaling pathway by luteinizing hormone in trout theca layers. Mol Cell Endocrinol. 2003, 205 (1-2): 11-20. 10.1016/S0303-7207(03)00208-9.
Montserrat N, González A, Méndez E, Piferrer F, Planas JV: Effects of follicle stimulating hormone on estradiol-17[beta] production and P-450 aromatase (CYP19) activity and mRNA expression in brown trout vitellogenic ovarian follicles in vitro. Gen Comp Endocrinol. 2004, 137 (2): 123-131. 10.1016/j.ygcen.2004.02.011.
Koskinen H, Pehkonen P, Vehniäinen E, Krasnov A, Rexroad C, Afanasyev S, Mölsa H, Oikari A: Response of rainbow trout transcriptome to model chemical contaminants. Biochem Biophys Res Commun. 2004, 320 (3): 745-753. 10.1016/j.bbrc.2004.06.024.
Krasnov A, Koskinen H, Pehkonen P, Rexroad CE, Afanasyev S, Molsa H: Gene expression in the brain and kidney of rainbow trout in response to handling stress. BMC Genomics. 2005, 6 (1): 3-10.1186/1471-2164-6-3.
Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, Harris MA, Hill DP, Issel-Tarver L, Kasarskis A, Lewis S, Matese JC, Richardson JE, Ringwald M, Rubin GM, Sherlock G: Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet. 2000, 25 (1): 25-29. 10.1038/75556.
Livak KJ, Schmittgen TD: Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2(-Delta DeltaC(T)) Method. Methods. 2001, 25 (4): 402-408. 10.1006/meth.2001.1262.
MacKenzie S, Balasch JC, Novoa B, Ribas L, Roher N, Krasnov A, Figueras A: Comparative analysis of the acute response of the trout, O. mykiss, head kidney to in vivo challenge with virulent and attenuated infectious hematopoietic necrosis virus and LPS-induced inflammation. BMC Genomics. 2008, 9: 141-10.1186/1471-2164-9-141.
MacKenzie S, Iliev D, Liarte C, Koskinen H, Planas JV, Goetz FW, Molsa H, Krasnov A, Tort L: Transcriptional analysis of LPS-stimulated activation of trout (Oncorhynchus mykiss) monocyte/macrophage cells in primary culture treated with cortisol. Mol Immunol. 2006, 43 (9): 1340-1348. 10.1016/j.molimm.2005.09.005.
Djordjevic B, Skugor S, Jorgensen SM, Overland M, Mydland LT, Krasnov A: Modulation of splenic immune responses to bacterial lipopolysaccharide in rainbow trout (Oncorhynchus mykiss) fed lentinan, a beta-glucan from mushroom Lentinula edodes. Fish Shellfish Immunol. 2009, 26 (2): 201-209. 10.1016/j.fsi.2008.10.012.
Mahajan KN, Mitchell BS: Role of human Pso4 in mammalian DNA repair and association with terminal deoxynucleotidyl transferase. Proc Natl Acad Sci. 2003, 100 (19): 10746-10751. 10.1073/pnas.1631060100.
Massard C, Zermati Y, Pauleau AL, Larochette N, Metivier D, Sabatier L, Kroemer G, Soria JC: hTERT: a novel endogenous inhibitor of the mitochondrial cell death pathway. Oncogene. 2006, 25 (33): 4505-4514. 10.1038/sj.onc.1209487.
Bobe J, Goetz FW: A novel osteopontin-like protein is expressed in the trout ovary during ovulation. FEBS Lett. 2001, 489 (2-3): 119-124. 10.1016/S0014-5793(01)02090-7.
Garczynski MA, Goetz FW: Molecular characterization of a ribonucleic acid transcript that is highly up-regulated at the time of ovulation in the brook trout (Salvelinus fontinalis) ovary. Biol Reprod. 1997, 57 (4): 856-864. 10.1095/biolreprod57.4.856.
Hajnik CA, Goetz FW, Hsu SY, Sokal N: Characterization of a ribonucleic acid transcript from the brook trout (Salvelinus fontinalis) ovary with structural similarities to mammalian adipsin/complement factor D and tissue kallikrein, and the effects of kallikrein-like serine proteases on follicle contraction. Biol Reprod. 1998, 58 (4): 887-897. 10.1095/biolreprod58.4.887.
Wada S, Kudo T, Kudo M, Sakuragi N, Hareyama H, Nishihira J, Fujimoto S: Induction of macrophage migration inhibitory factor in human ovary by human chorionic gonadotrophin. Hum Reprod. 1999, 14 (2): 395-399. 10.1093/humrep/14.2.395.
Bobe J, Goetz FW: A tumor necrosis factor decoy receptor homologue is up-regulated in the brook trout (Salvelinus fontinalis) ovary at the completion of ovulation. Biol Reprod. 2000, 62 (2): 420-426. 10.1095/biolreprod62.2.420.
Murdoch WJ, Van Kirk EA, Murdoch J: Plasmin cleaves tumor necrosis factor alpha exodomain from sheep follicular endothelium: implication in the ovulatory process. Biol Reprod. 1999, 60 (5): 1166-1171. 10.1095/biolreprod60.5.1166.
Terranova PF: Potential roles of tumor necrosis factor-alpha in follicular development, ovulation, and the life span of the corpus luteum. Domest Anim Endocrinol. 1997, 14 (1): 1-15. 10.1016/S0739-7240(96)00094-X.
Loret de Mola JR, Goldfarb JM, Hecht BR, Baumgardner GP, Babbo CJ, Friedlander MA: Gonadotropins induce the release of interleukin-1 beta, interleukin-6 and tumor necrosis factor-alpha from the human preovulatory follicle. Am J Reprod Immunol. 1998, 39 (6): 387-390.
Zolti M, Meirom R, Shemesh M, Wollach D, Mashiach S, Shore L, Rafael ZB: Granulosa cells as a source and target organ for tumor necrosis factor-alpha. FEBS Lett. 1990, 261 (2): 253-255. 10.1016/0014-5793(90)80565-Z.
Okuda K, Sakumoto R: Multiple roles of TNF super family members in corpus luteum function. Reprod Biol Endocrinol. 2003, 1: 95-10.1186/1477-7827-1-95.
Terranova PF, Rice VM: Review: cytokine involvement in ovarian processes. Am J Reprod Immunol. 1997, 37 (1): 50-63.
Wang HG, Takayama S, Rapp UR, Reed JC: Bcl-2 interacting protein, BAG-1, binds to and activates the kinase Raf-1. Proc Natl Acad Sci. 1996, 93 (14): 7063-7068. 10.1073/pnas.93.14.7063.
Flaws JA, Hirshfield AN, Hewitt JA, Babus JK, Furth PA: Effect of bcl-2 on the primordial follicle endowment in the mouse ovary. Biol Reprod. 2001, 64 (4): 1153-1159. 10.1095/biolreprod64.4.1153.
Pham CG, Bubici C, Zazzeroni F, Papa S, Jones J, Alvarez K, Jayawardena S, De Smaele E, Cong R, Beaumont C, Torti FM, Torti SV, Franzoso G: Ferritin heavy chain upregulation by NF-kappaB inhibits TNFalpha-induced apoptosis by suppressing reactive oxygen species. Cell. 2004, 119 (4): 529-542. 10.1016/j.cell.2004.10.017.
Curry TE, Smith MF: Impact of extracellular matrix remodeling on ovulation and the folliculo-luteal transition. Semin Reprod Med. 2006, 24 (4): 228-241. 10.1055/s-2006-948552.
Berndtson AK, Goetz FW: Protease activity in brook trout (Salvelinus fontinalis) follicle walls demonstrated by substrate-polyacrylamide gel electrophoresis. Biol Reprod. 1988, 38 (2): 511-516. 10.1095/biolreprod38.2.511.
Berndtson AK, Goetz FW: Metallo-protease activity increases prior to ovulation in brook trout (Salvelinus fontinalis) and yellow perch (Perca flavescens) follicle walls. Biol Reprod. 1990, 42 (2): 391-398. 10.1095/biolreprod42.2.391.
Coffman MA, Pinter JH, Goetz FW: Trout ovulatory proteins: site of synthesis, regulation, and possible biological function. Biol Reprod. 2000, 62 (4): 928-938. 10.1095/biolreprod62.4.928.
Bobe J, Montfort J, Nguyen T, Fostier A: Identification of new participants in the rainbow trout (Oncorhynchus mykiss) oocyte maturation and ovulation processes using cDNA microarrays. Reprod Biol Endocrinol. 2006, 4: 39-10.1186/1477-7827-4-39.
Ogiwara K, Takano N, Shinohara M, Murakami M, Takahashi T: Gelatinase A and membrane-type matrix metalloproteinases 1 and 2 are responsible for follicle rupture during ovulation in the medaka. Proc Natl Acad Sci. 2005, 102 (24): 8442-8447. 10.1073/pnas.0502423102.
Carnevali O, Cionna C, Tosti L, Lubzens E, Maradonna F: Role of cathepsins in ovarian follicle growth and maturation. Gen Comp Endocrinol. 2006, 146 (3): 195-203. 10.1016/j.ygcen.2005.12.007.
Walters KA, Allan CM, Handelsman DJ: Androgen actions and the ovary. Biol Reprod. 2008, 78 (3): 380-389. 10.1095/biolreprod.107.064089.
Fitzpatrick MS, Kraak Van der G, Schreck CB: Profiles of plasma sex steroids and gonadotropin in coho salmon, Oncorhynchus kisutch, during final maturation. Gen Comp Endocrinol. 1986, 62 (3): 437-451. 10.1016/0016-6480(86)90054-7.
Crespo D, Planas JV, Bobe J: Lipopolysaccharide administration in preovulatory rainbow trout (Oncorhynchus mykiss) reduces egg quality. Aquaculture. 2010, 300 (1-4): 240-242.
Bobe J, Goetz FW: Molecular cloning and expression of a TNF receptor and two TNF ligands in the fish ovary. Comp Biochem Physiol. 2001, 129 (2-3): 475-481. 10.1016/S1096-4959(01)00353-0.
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.
The authors declare that they have no competing interests.
DC performed gene expression analyses, testosterone measurement, follicle contraction determinations and the writing of the manuscript. EB participated in the generation of the apoptosis and cell viability data. NR carried out the generation of recombinant TNFα. SM participated in the design of the study. AK participated in the microarray analysis. FWG participated in the design of the study and in the execution of the LPS experiment. JB participated in the design of the study, in execution of the experiments and in the writing of the manuscript. JVP coordinated and participated in the design of the study as well as in the execution of the experiments and drafted the manuscript. All authors read and approved the final manuscript.
Authors’ original submitted files for images
Below are the links to the authors’ original submitted files for images.
About this article
Cite this article
Crespo, D., Bonnet, E., Roher, N. et al. Cellular and molecular evidence for a role of tumor necrosis factor alpha in the ovulatory mechanism of trout. Reprod Biol Endocrinol 8, 34 (2010). https://doi.org/10.1186/1477-7827-8-34
- Granulosa Cell
- Brown Trout
- Ovulatory Process
- Log2 Expression Ratio
- Follicle Wall