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E-GEOD-20296 - Cellular and molecular evidence for a role of tumor necrosis factor alpha in the ovulatory mechanism of trout

Status
Submitted on 12 February 2010, released on 14 May 2010, last updated on 1 May 2014
Organism
Oncorhynchus mykiss
Samples (2)
Array (1)
Protocols (7)
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 α (TNFα) and other immune factors, are produced and act on the reproductive system. However, TNFα 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 TNFα in vitro in preovulatory ovarian follicles of a teleost fish, the brown trout (Salmo trutta). In control and recombinant trout TNFα (rtTNFα)-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 rtTNFα on follicle contraction and testosterone production in preovulatory trout ovarian follicles. In addition, we analyzed the gene expression profiles of control and rtTNFα-treated ovarian tissue by microarray and real-time PCR (qPCR) analyses.Treatment with rtTNFα 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, rtTNFα causes a significant increase in follicle contraction and testosterone production. Also, using a salmonid-specific microarray platform (SFA2.0 immunochip) we observed that rtTNFα induces the expression of genes known to be involved in inflammation, proteolysis and tissue remodeling. In view of these results, we propose that TNFα 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 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. 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. 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 15ºC 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 -80ºC until assayed.
Experiment type
transcription profiling by array 
Contacts
Aleksei Krasnov <aleksei.krasnov@nofima.no>, Diego Crespo, Emilie Bonnet, Frederick W Goetz, Josep V Planas, Julien Bobe, Nerea Roher, Simon MacKenzie
MIAME
PlatformsProtocolsVariablesProcessedRaw
Files
Investigation descriptionE-GEOD-20296.idf.txt
Sample and data relationshipE-GEOD-20296.sdrf.txt
Raw data (1)E-GEOD-20296.raw.1.zip
Processed data (1)E-GEOD-20296.processed.1.zip
Array designA-GEOD-10092.adf.txt
Links