Amphibian eggs are polarized along an animal-vegetal (A-V) axis defined by the asymmetric distribution of pigment and yolk, and by the location of the meiotic spindles. The A-V axis formed during oogenesis plays an important role in the establishment of the germ layers during later embryonic development. Microtubules and microtubule organizing centers have often been implicated in specification of the A-V axis, though the precise mechanism remains unknown. The proposed experiments are designed to determine the organization and dynamics of microtubules and microtubule organizing centers during oogenesis and A-V axis formation in Xenopus laevis. Confocal immunofluorescence microscopy will be used to examine the organization of total and acetylated (non- dynamic) microtubules during oogenesis. Microinjection of biotinylated-tubulin will be used to characterize the microtubule assembly sites and dynamics in stage I oocytes, during A-V axis formation in stages III-IV, and during oocyte maturation. Microinjection of fluorescein-labeled tubulin and time-lapse confocal microscopy will be used to examine microtubule dynamics in living stage I oocytes and during assembly and alignment of the meiotic spindles. %%% The oocyte of Xenopus laevis is a large spherical cell that exhibits a nonrandom distribution of cytoplasmic components early in its development. The mechanism that controls this redistribution is not known, but is significant because it underlies polarized growth of the embryo. This project will provide important information on the roles of microtubules and microtubule organizing centers on the formation of the animal- vegetal axis of this cell.
