Tubulin and microtubule‐associated protein pools in unfertilized and fertilized eggs of the trout Oncorhynchus mykiss

Abstract

A molecular characterization of tubulin and microtubule‐associated proteins (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 obtained by cycles of assembly‐disassembly and by taxol‐induced polymerization, thus allowing identification of the protein components of isolated microtubules from the oocyte. Extraction procedures were developed in order to separate molecular components of the egg vitelum prior to purification steps. The use of antibodies that specifically tag tubulin and a set of site‐directed probes against repetitive binding sequences on MAPs provided data on the presence of tubulins and enabled the identification of an 85‐kDa protein that shares common functional epitopes with mammalian MAPs. An enzyme‐linked immunosorbent assay analysis 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 known effects of heat shock in oocyte fertilization, temperature changes from 4 to 26.5°C of fertilized eggs resulted in a transient increase in the soluble tubulin pools during the initial 5‐min heat incubation, decaying after 10 min treatment, to reach at 15 min the levels 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 the specific immunological probes. In contrast to tubulin no major differences were found between free MAPs pools of the fertilized oocytes as compared with unfertilized controls. However, heat shock treatment of fertilized oocytes also induced a transient increase in free MAP pools during the first 5 min followed by a mobilization of immunoreactive MAP components from the soluble to the assembled pools