Fig. 7. Methyl-ß-cyclodextrin (MCD) inhibits tyrosine kinase signaling and egg activation in fertilized Xenopus eggs. (A) Xenopus unfertilized eggs were pretreated with 1x DB containing the indicated concentrations of MCD for 60 minutes, washed and inseminated. Appearance of the first cleavage furrow in the eggs was monitored for 100 minutes after insemination. More than 50 eggs were examined in each MCD concentration. (B) LD-DIM fraction was prepared from unfertilized eggs that had been treated with or without MCD as in A. The samples (50 µl each) were subjected to protein kinase assay and analyzed by immunoblotting with anti-phosphotyrosine antibody (PY99, 1 µg/ml). Asterisks indicate the positions of bands that disappear by high dose of MCD. (C) Xenopus unfertilized eggs were pretreated with or without 25 mM MCD for 60 minutes and then left untreated (Uf), or activated by either fertilization (F, 5 minutes of insemination) or H2O2 (H, 10 mM, 5 minutes). LD-DIM fractions (20 µl per lane) were obtained from the egg samples and analyzed for their protein profile by silver staining. (D) LD-DIM fractions (20 µl each) prepared as in C were analyzed by immunoblotting with anti-phosphotyrosine antibody. Asterisks indicate the positions of bands that disappear in the presence of MCD. (E) Eggs were injected with fura-2 and then pretreated with or without 25 mM MCD for 60 minutes. After the pretreatment, eggs were inseminated while recording the fluorescent ratio signal using a high-frame digital CCD imaging ARGUS/HISCA system (Hamamatsu Photonics, Japan). Shown are traces of the fura-2 signal as a function of time. Black arrowheads indicate the time at which insemination was started. In some experiments, A23187 (0.5 µM) was applied after 30 minutes of insemination of MCD-treated eggs (white arrohead). (F) LD-DIM fractions were prepared from untreated and MCD (25 mM)-treated unfertilized eggs and analyzed by immunoblotting with either anti-Xyk (100x dilution of antiserum), anti-integrin ß1(100x dilution of antiserum), anti-Ras (0.5 µg/ml) or anti-Gq
(2 µg/ml) antibody. (G) LD-DIM fractions of untreated and MCD-treated eggs prepared as in F were assayed for their relative cholesterol content. The cholesterol content in the untreated egg LD-DIM was taken as 100%. Data represent mean±standard deviations of three independent trials. A bar with an asterisk is significantly different from the control. P <0.01.
