Project description: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 Î± (TNFÎ±) and other immune factors, are produced and act on the reproductive system. However, TNFÎ± 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 TNFÎ± in vitro in preovulatory ovarian follicles of a teleost fish, the brown trout (Salmo trutta). In control and recombinant trout TNFÎ± (rtTNFÎ±)-treated 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 rtTNFÎ± on follicle contraction and testosterone production in preovulatory trout ovarian follicles. In addition, we analyzed the gene expression profiles of control and rtTNFÎ±-treated ovarian tissue by microarray and real-time PCR (qPCR) analyses.Treatment with rtTNFÎ± induces ovarian 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, rtTNFÎ± causes a significant increase in follicle contraction and testosterone production. Also, using a salmonid-specific microarray platform (SFA2.0 immunochip) we observed that rtTNFÎ± induces the expression of genes known to be involved in inflammation, proteolysis and tissue remodeling. In view of these results, we propose that TNFÎ± 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. Reproductively mature female brown trout (Salmo trutta) from a cultured stock at the Piscifactoria de BagÃ (Generalitat de Catalunya, BagÃ , 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. After dissection, brown trout preovulatory ovaries were placed in HankÂ´s balanced salt solution (HBSS) and individual ovarian follicles were manually separated with forceps from each ovary on ice, as previously described. 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 rtTNFÎ± (100 ng/ ml, dissolved directly in HBSS-BSA), at 15ÂºC 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 -80ÂºC until assayed.

Project description:The proliferative darkening syndrome (PDS) is an annually recurring disease that causes species-specific die-off of brown trout (Salmo trutta fario) in PDS-impacted pre-alpine rivers of central Europe. The mortality rate for PDS is near 100% and consequently the survival of brown trout populations in the impacted regions is threatened. The progression of PDS occurs in two stages, a subclinical stage and a symptomatic stage, over a time period of more than 3 months starting in spring and culminating with the die-off of brown trout in late summer. Based on experimental evidence it is hypothesized that PDS is caused by an infectious agent. To substantiate this working hypothesis as well as to discern the type of pathogen likely to be responsible for PDS, microarray analysis were conducted using a salmonid-specific cDNA microarray to assess the hepatic immune response of brown trout during the progression of PDS in 7-day intervals over a time period of 98 days.The microarray analysis revealed that brown trout suffering from PDS exhibit increased hepatic expression of important anti-viral genes both during the subclinical stage of PDS, namely the Barrier-to-autointegration factor, and during the symptomatic stage of PDS, namely the interferon regulatory factor 1 as well as the Guanylate-binding protein 1. Additionally, during the symptomatic stage of PDS there is strong hepatic up-regulation of the chemokine CCL19, complement components C1QC and C6, Proteasome activator complex subunits 1 and 2 as well as a variety of both MHC class I and MHC class II transcripts. In conclusion, the increased expression of hepatic immune response genes involved in a variety of immune system processes that mediate defense against infection identified by the microarray analysis during the progression of PDS support the working hypothesis that PDS is caused by an infectious agent. Furthermore, the up-regulation of antiviral immune response genes during both the subclinical stage and the symptomatic stage of PDS suggests that this disease is caused by a pathogenic virus. On May 29, 2008, brown trout (Salmo trutta fario) of the same age class (1+) with an individual weight ranging between 25-85 grams were obtained from a single hatchery (Schwäbischer Fischereihof Salgen, Fachberatung für Fischerei Schwaben, Germany) and randomly allocated to one of two different experimental stations that are both located along the Iller river, named here simply the control station (location by Oberstdorf, Germany) and the treatment station (location by Kempten, Germany). At both experimental stations brown trout were held in tanks (n=750 at the treatment station and n=70 at the control station) that were supplied with water from the Iller river in a flow-through system. Sampling at both experimental stations started on May 29, 2008, which was also the day on which the fish were first transferred to their exposure tanks (referred to as 0 day post exposure; d.p.e), and ended on the 5th of September 2008. At the treatment station 3 fish were sampled each day (always at 2pm), whereas at the control station 3 fish were sampled in 7-day intervals (always at 12 noon). Fish were anaesthetized by a blow to the head and organ tissue of interest (liver, kidney, spleen, gill, muscle, stomach and foregut tissue) were immediately harvested from sacrificed fish, snap-frozen in liquid nitrogen and subsequently stored at -80°C. Livers from the three brown trout (n=3) that were sampled concurrently from both treatment and control group (n=2) in 7-day intervals starting 7 d.p.e (7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91 and 98 d.p.e; n=14) were recruited for the use in the microarray analysis. Microarray analyses were performed using a direct comparison two-channel design in which equimolar amounts of liver sample of one brown trout from both treatment and control group were co-hybridized on the same microarray. For each time point (n=14) microarray co-hybridizations were repeated in triplicate (n=3) and included one dye-swap in order to reduce dye-bias. Thus a total of 42 microarray slides were used in this study (3 biological replicate microarrays x 14 time points = 42 microarrays).

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