Unknown,Transcriptomics,Genomics,Proteomics

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Luteinizing hormone induces ovulation via tumor necrosis factor Î±-dependent increases in prostaglandin F2Î± in a nonmammalian vertebrate

ABSTRACT: Ovulation is induced by the preovulatory surge of luteinizing hormone (LH) that acts on the ovary and triggers the rupture of the preovulatory ovarian follicle by stimulating proteolysis and apoptosis in the follicle wall, causing the release of the mature oocyte. In mammals, the pro-inflammatory cytokine tumor necrosis factor Î± (TNFÎ±) and prostaglandin F2Î± (PGF2Î±) are known to be involved in the control of ovulation but their role mediating the pro-ovulatory actions of LH has not been established. Here we show that Lh induces PGF2Î± synthesis through its stimulation of TnfÎ± production in trout, a primitive teleost fish. Importantly, trout recombinant TnfÎ± (rTnfÎ±) and PGF2Î± recapitulate the stimulatory in vitro effects of salmon Lh (sLh) on contraction, proteolysis and loss of cell viability in the preovulatory follicle wall and, finally, ovulation. Furthermore, all pro-ovulatory actions of sLh are blocked by inhibition of TnfÎ± secretion or PG synthesis and those of rTnfÎ± are blocked by PG synthesis inhibitors. Therefore, we provide evidence that the TnfÎ±âdependent increase in PGF2Î± production is necessary for the pro-ovulatory actions of Lh in a teleost fish. The results from this study shed light onto the mechanisms underlying the pro-ovulatory actions of LH in vertebrates and may prove important in clinical assessments of female infertility. Preovulatory follicles from brook trout from four different females (n = 4) were isolated and incubated in the absence or presence of salmon LH. Total RNA of control and LH-treated preovulatory follicles from each of the four females was analyzed.

ORGANISM(S): Salmo trutta

SUBMITTER: Josep Planas

PROVIDER: E-GEOD-67592 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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:Gonadotropin surge acts on the preovulatory follicle of the ovary to induce luteinization of follicular cells, oocyte meiotic maturation, cumulus expansion and follicular rupture leading to ovulation. These processes are brought about by spatial and temporal changes in transcriptional regulation of genes in the follicular cells in response to the gonadotropin surge. Analysis of gene expression changes in the periovulatory follicular cells will help in delineating the signal transduction pathways involved in the above mentioned processes. In monoovulatory species like bovines, the time interval of 24-28 hours between gonadotropin surge and ovulation provides distinct advantage for studying the temporal changes in the gene expression pattern. Thus, in the present study, we attempt to identify the temporal changes in the global gene expression profile in the periovulatory follicle of buffalo cows in response to gonadotropin surge and the results suggest the involvement of Insulin-like Growth Factor 1 and cytokine signaling pathways in the periovulatory events. Experiment Overall Design: To study the periovulatory gene expression changes in buffalo cows, an induced-ovulation model system involving sequential treatment with PGF2alpha and GnRH was standardized. The follicular wave containing at least one large follicle of ~7mm size was determined by ultrasonography on day 7 of the estrous cycle before administering exogenous PGF2alpha to induce luteolysis and follicular growth. Exogenous GnRH (100Âµg i.m) was administered 36h post PGF2alpha to induce LH surge. The time course of increase in LH levels post GnRH injection was monitored. Since peak LH levels are attained 2 h post GnRH administration, the time intervals of 3 h post GnRH (corresponding to1 h post LH surge) and 24 h post GnRH (corresponding to 22 h post LH surge) were chosen to identify the gene expression profile associated with immediate early and delayed changes in periovulatory follicle respectively. Thus ovaries were collected before, 1 h and 22 h post LH surge and follicle wall and granulosa cells were isolated from the ovaries and snap frozen for the purpose of RNA isolation.

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Luteinizing hormone induces ovulation via tumor necrosis factor Î±-dependent increases in prostaglandin F2Î± in a nonmammalian vertebrate

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