TUBULIN AND MICROTUBULE-ASSOCIATED PROTEIN POOLS IN UNFERTILIZED AND FERTILIZED-EGGS OF THE TROUT ONCORHYNCHUS-MYKISS

A molecular characterization of tubulin and microtubule-associated pro teins (MAPs) along with their intracellular pool distributions in both unfertilized and fertilized oocytes of the trout Oncorhynchus mykiss was carried out. In vitro assembly of microtubular proteins was obtain ed by cycles of assembly-disassembly and by taxol-induced polymerizati on, thus allowing identification of the protein components of isolated microtubules from the trout oocyte. Extraction procedures were develo ped in order to separate molecular components of the egg vitelum prior to purification steps. The use of antibodies that specifically tag tu bulin and a set of site-directed probes against repetitive binding seq uences on MAPs provided data on the presence of tubulins and enabled t he identification of an 85-kDa protein that shares common functional e pitopes with mammalian MAPs. An enzyme-linked immunosorbent assay anal ysis of the free soluble tubulin pools revealed a significant decrease in the pool extent during fertilization as compared with unfertilized oocytes controls. Interestingly, this decrease in free tubulin in the fertilized trout oocyte appeared to be accompanied with a concomitant increase of the assembled tubulin pools. Within the context of the kn own effects of heat shock in oocyte fertilization, temperature changes from 4 to 26.5 degrees C of fertilized eggs resulted in a transient i ncrease in the soluble tubulin pools during the initial 5-min heat inc ubation, decaying after 10 min treatment, to reach at 15 min the level s of soluble tubulin pools of untreated controls. Total tubulin pools remained constant during the heat incubations of fertilized eggs. The distribution of MAPs pools in the oocyte was also investigated using t he specific immunological probes. In contrast to tubulin no major diff erences were found between free MAPs pools of the fertilized oocytes a s compared with unfertilized controls. However, heat shock treatment o f fertilized oocytes also induced a transient increase in free MAP poo ls during the first 5 min followed by a mobilization of immunoreactive MAP components from the soluble to the assembled pools. (C) 1995 Wile y-Liss, Inc.