Confocal microscopy and 3‐D reconstruction of the cytoskeleton of Xenopus oocytes

Abstract

Xenopus oocytes contain a complex cytoskeleton composed of three filament systems: (1) microtubules, composed of tubulin and at least three different microtubule-associated proteins (XMAPs); (2) microfilaments composed of actin and associated proteins; and (3) intermediate filaments, composed of keratins. For the past several years, we have used confocal immunofluorescence microscopy to characterize the organization of the oocyte cytoskeleton throughout the course of oogenesis. Together with computer-assisted reconstruction of the oocyte in three dimensions, confocal microscopy gives an unprecedented view of the assembly and reorganization of the cytoskeleton during oocyte growth and differentiation. Results of these studies, combined with the effects of cytoskeletal inhibitors, suggest that organization of the cytoskeleton in Xenopus oocytes is dependent upon a hierarchy of interactions between microtubules, microfilaments, and keratin filaments. This article presents a gallery of confocal images and 3-D reconstructions depicting the assembly and organization of the oocyte cytoskeleton during stages 0-VI of oogenesis, a discussion of the mechanisms that might regulate cytoskeletal organization during oogenesis, and speculates on the potential roles of the oocyte cytoskeleton during oogenesis and axis formation.

Water and lipid MRI of the Xenopus oocyte

“…With a mean length of ϳ600 m, approximately 0.5-1 million MTs form a network in the cytoplasm (11). A Stage VI oocyte also contains 1.72 g of actin (12), and cables of F-actin span the nucleus as well as the cytoplasm (11). The nucleus and/or cytoplasm also house other proteins, nucleic acids, and a myriad of small organic molecules.…”

Apparent mitochondrial asymmetry in Xenopus eggs

“…One of the first manifestations of polarity in the developing Xenopus oocyte is the presence of a mitochondrial cloud within the vegetal hemisphere of immature (stage I; Dumont, 1972) oocytes (Heasman et al, 1984). Subsequently, dynamic reorganization of the cytoskeleton (Gard, 1999) and localization of specific mRNAs and protein (reviewed in Mowry and Cote, 1999) occur during defined periods during growth of the oocyte, resulting in molecular asymmetry along the AV axis of the fully grown oocyte. By the end of oogenesis, polarity is also externally evident, with pigment restricted to the animal hemisphere and yolk platelets being larger and more abundant in the vegetal hemisphere (Danilchik and Gerhart, 1987).…”

Organization of cytokeratin cytoskeleton and germ plasm in the vegetal cortex of Xenopus laevis oocytes depends on coding and non-coding RNAs: Three-dimensional and ultrastructural analysis

“…The vegetal cortex contains elements of cytoskeleton such as microtubules, actin and cytokeratin filaments (which anchor various localized RNAs), cortical granules, yolk platelets and germ plasm islands containing germ line mitochondria and germinal granules (17,(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29). Although there is a substantial volume of data on the ultrastructure of the vegetal cortex in Xenopus oocytes, and on the changes occurring in the cortex during oocyte maturation and egg activation (19,30, 31; reviewed in 20), the information on the organization of the cytokeratin cytoskeleton in Xenopus oocytes and eggs comes mainly from light and confocal microscopy (17,19,24,32; reviewed in 20) and so far, there is no detailed information on the ultrastructural organization of the cytokeratin in relation to other components of the cortex such as the yolk, cortical granules and germ plasm.…”