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.