M4 PM Endocrine Disruption in Fish (Part 2)
Monday, 14 November 2005: 1:50 PM - 5:30 PM in Ballroom 4

(ROY-1117-829614) Vitellogenin levels in three-spine sticklebacks (Gasterosteus aculeatus) to detect effects of pesticide exposure.

Roy, R.¹, Maltais, D.¹, Couillard, C.¹, Lebeuf, M.¹, ¹ Fisheries and Oceans Canada, Mont-Joli, Quebec, Canada

ABSTRACT- As part of a project investigating effects of pesticides on fish, we isolated vitellogenin (VTG) from threespine sticklebacks (Gasterosteus aculeatus). In June - July 2003, sticklebacks were captured from a site on the South shore of the St Lawrence Estuary (SLE). In the laboratory, mature females (n = 120) were anaesthetised, injected with a proteolysis inhibitor (aprotinin) and sampled for blood. Plasma was obtained by centrifugation. The protein was precipitated from plasma with MgCl₂and EDTA, following modifications of the method described by Norberg (1995) and further purified by gel filtration. The purity of the pooled VTG fractions was confirmed by Native-PAGE (3-8% tris-acetate gel). Molecular weight (MW) was determined by gel filtration run with MW standards. Stickleback VTG has a relatively high MW of approximately 637 kDa (vs 520 kDa for smooth flounder VTG, Roy et al. 2004). Precipitated plasma from mature female sticklebacks and the presumptive VTG stained with Coomassie blue, suggesting that the latter is a female-specific protein. Staining with methyl green (phosphoprotein), Sudan black B (lipoprotein, Prat et al. 1969) and Periodic Acid-Schiff reagent (glycoprotein) on Native-PAGE identifies the purified protein as a phospho-lipo-glycoprotein - further evidence that it is VTG. Finally, Western blot with commercially available antibodies confirmed that the protein is stickleback VTG. We developed a homologous enzyme immunoassay for stickleback VTG, using the purified protein for standards, and analysed plasma from mature sticklebacks from 3 other SLE sites. The results suggest that VTG levels in these populations are not affected by pesticides.

Key words: vitellogenin, stickleback, EIA, pesticides