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• Comparison of exposure of zebrafish to ethinylestradiol and estetrol on fecundity. • Ethinylestradiol significantly impacted zebrafish reproductive parameters. • Few significant effects were reported with estetrol on zebrafish reproduction. • Estetrol presents a more favorable environmental profile than ethinylestradiol. Natural and synthetic oestrogens are commonly found in aquatic ecosystems. The synthetic oestrogen 17α-ethinylestradiol (EE2) is widely used in oral contraceptives and its ecotoxicological effects on aquatic organisms have been widely reported. The natural oestrogen estetrol (E4) was recently approved for use in a new combined oral contraceptive and, after therapeutic use, is likely to be found in the aquatic environment. However, its potential effects on non-target species such as fish is unknown. In order to characterize and compare the endocrine disruptive potential of E4 with EE2, zebrafish (Danio rerio) were exposed to E4 or EE2 in a fish short-term reproduction assay conducted according to OECD Test Guideline 229. Sexually mature male and female fish were exposed to a range of concentrations, including environmentally relevant concentrations of E4 and EE2, for 21 days. Endpoints included fecundity, fertilization success, gonad histopathology, head/tail vitellogenin concentrations, as well as transcriptional analysis of genes related to ovarian sex steroid hormones synthesis. Our data confirmed the strong impact of EE2 on several parameters including an inhibition of fecundity, an induction of vitellogenin both in male and female fish, an alteration of gonadal structures and the modulation of genes involved in sex steroid hormone synthesis in female fish. In contrast, only few significant effects were observed with E4 with no impact on fecundity. The results suggest that the natural oestrogen, E4, presents a more favorable environmental profile than EE2 and is less likely to affect fish reproductive capacity. [ABSTRACT FROM AUTHOR]

Platelets are small blood cells derived from cytoplasmic fragments of megakaryocytes and play an essential role in thrombosis and hemostasis. Platelet activation depends on the rapid phosphorylation and dephosphorylation of key signaling molecules, and a number of kinases and phosphatases have been identified as major regulators of platelet function. However, the investigation of novel signaling proteins has suffered from technical limitations due to the anucleate nature of platelets and their very limited levels of mRNA and de novo protein synthesis. In the past, experimental methods were restricted to the generation of genetically modified mice and the development of specific antibodies. More recently, novel (phospho)proteomic technologies and pharmacological approaches using specific small-molecule inhibitors have added additional capabilities to investigate specific platelet proteins.In this chapter, we report methods for using genetic and pharmacological approaches to investigate the function of platelet signaling proteins. While the described experiments focus on the role of the dual-specificity phosphatase 3 (DUSP3) in platelet signaling, the presented methods are applicable to any signaling enzyme. Specifically, we describe a testing strategy that includes (1) aggregation and secretion experiments with mouse and human platelets, (2) immunoprecipitation and immunoblot assays to study platelet signaling events, (3) detailed protocols to use selected animal models in order to investigate thrombosis and hemostasis in vivo, and (4) strategies for utilizing pharmacological inhibitors on human platelets.

Inflammation shifts the hemostatic mechanisms in favor of thrombosis. Upon tissue damage or infection, a sudden increase of extracellular ATP occurs, that might contribute to the crosstalk between inflammation and thrombosis. On platelets, P2X1 receptors act to amplify platelet activation and aggregation induced by other platelet agonists. These receptors critically contribute to thrombus stability in small arteries. Besides platelets, studies by our group indicate that these receptors are expressed by neutrophils. They promote neutrophil chemotaxis, both in vitro and in vivo. In a laser-induced injury mouse model of thrombosis, it appears that neutrophils are required to initiate thrombus formation and coagulation activation on inflamed arteriolar endothelia. In this model, by using P2X1-/ - mice, we recently showed that P2X1 receptors, expressed on platelets and neutrophils, play a key role in thrombus growth and fibrin generation. Intriguingly, in a model of endotoxemia, P2X1-/ - mice exhibited aggravated oxidative tissue damage, along with exacerbated thrombocytopenia and increased activation of coagulation, which translated into higher susceptibility to septic shock. Thus, besides its ability to recruit neutrophils and platelets on inflamed endothelia, the P2X1 receptor also contributes to limit the activation of circulating neutrophils under systemic inflammatory conditions. Taken together, these data suggest that P2X1 receptors are involved in the interplay between platelets, neutrophils and thrombosis. We propose that activation of these receptors by ATP on neutrophils and platelets represents a new mechanism that regulates thrombo-inflammation.