Confocal immunofluorescence microscopy of ovaries from juvenile frogs revealed changes in the organization, acetylation, and nucleation, of microtubules (MTs), and redistribution of gamma-tubulin (gamma-TB), during early oogenesis in Xenopus laevis. Interphase oogonia contained sparse, radially organized, MT arrays and prominent centrosomes, Acetylated MTs were not commonly found in oogonia. In contrast, small (approximately 12-25 microns), postmitotic (stage 0) oocytes contained dense, highly polarized, MT networks that exhibited little or no evidence of radical organization. Examination of stage 0 oocytes stained with antibodies to gamma-TB, in conjunction with assays of MT nucleation activity, revealed that stage 0 oocytes do contain active centrosomes. In addition, stage 0 oocytes contained numerous acetylated MTs, suggesting that arrest in meiotic prophase is accompanied by MT stabilization. Early stage I oocytes (diameters from approximately 35-50 microns) exhibited a rounded morphology and contained a dispersed, apparently disordered, MT array with a substantial population of acetylated MTs. Examination of stage I oocytes stained with gamma-TB antibodies revealed that this centrosomal protein was present in multiple cytoplasmic foci which did not function as MTOCs following cold-induced MT disassembly. The results presented indicate that the maternal centrosome is inactivated during early stage I, roughly coincident with the onset of the diplotene stage of meiotic prophase and prior to assembly of the mitochondrial mass. Our observations place constraints on the role of MTs and the maternal centrosome during specification of the animal-vegetal axis of Xenopus oocytes and raise questions regarding the mechanisms by which MT assembly and organization are regulated during oocyte differentiation.
We used affinity-purified polyclonal antibodies to characterize the distribution and function of XMAP230, a heat-stable microtubule-associated protein isolated from Xenopus eggs, during oogenesis. Immunoblots revealed that XMAP230 was present throughout oogenesis and early development, but was most abundant in late stage oocytes, eggs, and early embryos. Immunofluorescence microscopy revealed that XMAP230 was associated with microtubules in oogonia, post-mitotic stage 0 oocytes, early stage I oocytes, and during stage IV-VI of oogenesis. However, staining of microtubules by anti-XMAP230 was not detectable during late stage I through stage III. In stage VI oocytes, anti-XMAP230 stained a large subset of microtubules that were also stained with monoclonal antibodies specific for acetylated (α)-tubulin. During oocyte maturation, XMAP230 was associated with the transient microtubule array that serves as the precursor of the first meiotic spindle, as well as both first and second meiotic spindles. The extensive array of cytoplasmic microtubules present throughout maturation was not detectably stained by anti-XMAP230. Microinjection of anti-XMAP230 locally disrupted the organization and acetylation of microtubules in stage VI oocytes, and reduced the re-acetylation of microtubules during recovery from cold-induced microtubule disassembly. Subsequent maturation of oocytes injected with anti-XMAP230 resulted in defects in the assembly of the transient microtubules array and first meiotic spindle. These observations suggest that XMAP230 is required for the stabilization and organization of cytoplasmic and spindle microtubules in Xenopus oocytes and eggs.
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