Measuring Ca2+-signalling at fertilization in the sea urchin Psammechinus miliaris: Alterations of this Ca2+-signal by copper and 2,4,6-tribromophenol

Abstract

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 µM Cu 2+ and 1 µM 2,4,6-tribromphenol. Bisphenol A significantly inhibited fertilization success at 438 µM while phenanthrene had no effect up to 56 µM. 6.3 µM copper and 100 µM 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.

Role and mechanism of the AMPK pathway in waterborne Zn exposure influencing the hepatic energy metabolism of Synechogobius hasta

“…Generally, divalent metals can affect cellular calcium homeostasis and calcium signalling3738. In the present study, waterborne Zn exposure up-regulated the expression of Ca 2+ -dependent proteins, such as calpain and calreticulin (CRT)3940 (Supplementary Table 5).…”

Inhibition of multidrug/xenobiotic resistance transporter by MK571 improves dye (Fura 2) accumulation in crustacean tissues from lobster, shrimp, and isopod

“…In different vertebrates and in marine algae, the calcium chelating dye Fura 2 was proven to be a suitable substrate for MRP transporters (Manzini et al, 2008;Scherer et al, 2008). In sea urchin eggs MK571 improved dye loading with Fura 2, which facilitated calcium measurements during fertilization (Schäfer et al, 2009). Our results provide indirect evidence for the existence of MRP transporters in the crustacean brain where it may serve as an important defence system especially against toxic lipophilic and amphiphilic small compounds.…”

Impact of Endocrine‐Disrupting Chemicals on Reproductive Function in Zebrafish (Danio rerio)

“…; Schäfer et al. ). In accordance with this conclusion, exposing adult zebrafish to TBP for 6 weeks decreased the proportion of fertilized eggs (Haldén et al.…”