Measuring Ca2+-signalling at fertilization in the sea urchin Psammechinus miliaris: alterations of this Ca2+-signal by copper and 2,4,6-tribromophenol.
During fertilization, eggs undergo a temporary rise in the intracellular concentration of free Ca(2+) ions. Using the membrane permeable acetoxymethylester of the fluorescent calcium indicator dye Fura-2, Fura-2 AM, the Ca(2+)-signal at fertilization was not detectable in eggs of the sea urchin Psammechinus miliaris. However, after treatment of the eggs with Fura-2 AM in combination with MK571, an inhibitor for multidrug resistance associated proteins, clear Ca(2+)-signals at fertilization could be measured without microinjection of the dye. We used this methodology to detect possible alterations of Ca(2+)-signalling at fertilization by exposure of eggs to environmental pollutants. For this purpose, the heavy metal copper, the bromophenol 2,4,6-tribromophenol, the organic compound bisphenol A and the polycyclic aromatic hydrocarbon phenanthrene were tested for their potential to inhibit fertilization success of P. miliaris. Copper and 2,4,6-tribromophenol showed a dose-dependent effect on fertilization rates of P. miliaris and significantly inhibited fertilization at 6.3 microM Cu(2+) and 1 microM 2,4,6-tribromphenol. Bisphenol A significantly inhibited fertilization success at 438 microM while phenanthrene had no effect up to 56 microM. 6.3 microM copper and 100 microM 2,4,6-tribromophenol significantly increased the Ca(2+)-signal at fertilization. This alteration may contribute to the reduced fertilization rates of P. miliaris after exposure to copper and 2,4,6-tribromophenol.
Schäfer
Sabine
S
Alfred Wegener Institute for Polar and Marine Research in Helmholtz Association, Am Handelshafen 12, 27570 Bremerhaven, Germany.
Bickmeyer
Ulf
U
Koehler
Angela
A
D000818:Animals; D001559:Benzhydryl Compounds; D020013:Calcium Signaling / Q000187:drug effects*; D019327:Copper Sulfate / Q000633:toxicity*; D004305:Dose-Response Relationship, Drug; D005306:Fertilization / Q000187:drug effects*; D008856:Microscopy, Fluorescence; D010616:Phenanthrenes / Q000633:toxicity; D010636:Phenols / Q000633:toxicity*; D012617:Sea Urchins / Q000187:drug effects* / Q000378:metabolism; D013084:Sperm-Ovum Interactions / Q000187:drug effects; D013997:Time Factors; D014874:Water Pollutants, Chemical / Q000633:toxicity*; D000098826:Bisphenol A Compounds
D001559:Benzhydryl Compounds; D019327:Copper Sulfate; D010616:Phenanthrenes; D010636:Phenols; D014874:Water Pollutants, Chemical; C031181:phenanthrene; C006780:bisphenol A; D000098826:Bisphenol A Compounds; C004554:2,4,6-tribromophenol