Characterization and variations of organic parameters in teleost fish endolymph during day-night cycle, starvation and stress conditions.

The aim of the present work was to examine the modifications of the organic composition of fish endolymph under environmental conditions (day-night cycle, starvation and Cl2-stress) known to modify otolith growth. Endolymph electrophoretic patterns were compared. An antibody raised against the trout otolith organic matrix allowed examining the variations of organic matrix precursors in the endolymph under the above conditions. Western blot analysis showed bands around 60-80 kDa. A 50% decrease of immunolabelling was observed during the night whereas increases were seen after starvation (factor 3) or stress (factor 2) suggesting that these variations could be related to the organic matrix deposit. A factor retarding in vitro CaCO3 crystallization (FRC) was shown to co-precipitate with endolymph proteins and its apparent molecular mass (determined by measuring the activity after electro elution of gel electrophoresis) was estimated around 20 kDa. The FRC activity was stable during day-night cycle whereas it decreased by 70% and nearly 100% under starvation and stress respectively. These results suggest that the FRC, although retarding in vitro crystallization, plays a major role in the process of otolith calcification and that the decreases measured after starvation and stress are responsible for the decreases of the otolith growth. The variations of these two parameters (precursors and FRC) could contribute for the changes in the microstructure of the otolith.

Ca2+ release from intracellular stores by thapsigargin in sea urchin eggs: Relationship to larval development and relevance in egg activation.

Thapsigargin (Tg), an inhibitor of microsomal Ca2+ ATPase, is used as a tool to study the changes in Ca2+ sequestration in sea urchin eggs and their relationship to embryonic development. Micromolar amounts of Tg inhibit ATP-dependent Ca2+ sequestration in a dose-dependent and non-reversible manner, depending on the bulk of biological material used. IC5O values are 1 nmol/L and 1-10µmol/L, respectively, in the cortical Ca2+ stores (isolated cortices preparation) and in digitonin-permeabilized eggs, a preparation giving access to the deeper reticulum compartment. Micromolar Tg does not induce Ca2+ release from 45 Ca pre-loaded cortices but leads to a loss of 25% of the total Ca2+ content from the cortical area. Using microspectrofluorimetry of fura-2-loaded eggs, we found that 10 µmol/L Tg induced a moderate rise in cytosolic Ca2+ activity as compared with the fertilization-induced Ca2+ transient whether eggs were fertilized or not. Early events related to fertilization as, for example, elevation of the fertilization envelope, proton excretion and sustained increase of amino acid uptake, are triggered by 10µmol/L Tg but with a delayed onset relative to sperm-induced effects. The present findings indicate that although it triggers most fertilization-related events, Tg cannot be considered as a true mitotic agent in sea urchin eggs. When added after fertilization, Tg affects cleavage and the further embryonic development giving rise to abnormalities comparable to the animalized larvae obtained with other compounds responsible for the inhibition of reticular Ca2+ sequestration.