Incubation of Eggs and Oocytes in D ~

We have assayed various materials for their ability to induce aster formation by microinjection into unfertilized eggs of Xenopus laevis. We have found that purified basal bodies from Chlamydomonas reinhardtii and Tetrahymena pyriformis induce the formation of asters and irregular cleavage furrows within 1 h after injection. Other microtubule structures such as flagella, flagellar axonemes, cilia, and brain microtubules are completely ineffective at inducing asters or cleavage furrows in unfertilized eggs. When known amounts of sonicated Tetrahymena and Chlamydomonas preparations are injected into unfertilized eggs, 50% of the injected eggs show a furrowing response at approximately 3 cell equvalents for Chlamydomonas and 0.1 cell equivalent for Tetrahymena. These results are close to those expected if basal bodies were the effective astral-inducing agent in these cells. Other materials effective at inducing asters in unfertilized eggs, such as crude brain nuclei, sperm, and a particulate fraction from brain known to induce parthenogenesis in eggs of Rana pipiens, probably contain centrioles as the effective agent. Our experiments provide the first functional assay to indicate that centrioles play an active role in aster initiation. None of the injected materials effective in unfertilized eggs produced any observable response in fully grown oocytes. Oocytes and eggs were found to have equal tubulin pools as judged by colchicine-binding activity. Therefore, the inability of oocytes to form asters cannot be due to a lack of an organizing center or to a lack of tubulin. Experiments in which D20 was found to stimulate aster-like fibrous areas in eggs but not oocytes suggest that the inability of oocytes to form asters may be due to an inability of tubulin in oocytes to assemble. Asters are radially symmetric structures composed of microtubules which assemble at the poles of most mitotic animal cells (19). Recent advances in our understanding of microtubule assembly in vitro (4, 14, 15, 30) have stimulated a reinvestigation of the regulation of aster assembly (31, 33). Aster assembly could be regulated on two distinct levels: the polymerization of tubulin into microtubules, and the localization and organization of this assembly process to form the aster structure. The study of aster formation in a living cell may therefore provide us with an understanding of how microtubule assembly and organization is controlled in vivo. THE JOURNAL OF CELL BIOLOGY 9 VOLUME 67, 1975 9 pages 105-117 105 on July 9, 2017 jcb.rress. org D ow nladed fom In experiments designed to determine the effect of the surrounding cytoplasm on nuclear structure and synthetic activity, Gurdon (10) injected nuclei into immature , fully grown oocytes and mature oocytes (unfertilized eggs) of Xenopus laevis. He observed that the nuclei injected into fully grown oocytes swell and synthesize RNA, while nuclei injected into mature oocytes condensed their chromosomes and formed mul t ipolar spindles. The appearance of spindles in mature oocytes but not in immature oocytes in response to injected nuclei suggested that these cells differ in their abiiity to form asters. We have extended the work of Gurdon, using the Xenopus oocyte and egg system for two purposes. First, we have utilized microinject ion in Xenopus eggs to assay mater ia ls for their ability to induce asters and we have tried to define the role of the centriole as such an inducing material . Second, we have made a prel iminary a t tempt to unders tand what underlies the difference in the capacity of oocytes and eggs to form asters. Mailer and Gerha r t have also explored the injection of centr iole-containing preparat ions and will report their results elsewhere. (J. Mai ler and J. Gerhar t . Unpubl ished observat ions) M A T E R I A L S A N D M E T H O D S