Confocal microscopy of cytoskeletal organization in amphibian oocytes and eggs

Abstract

Amphibian oocytes, eggs, and early embryos have been increasingly used as models for studying the regulation of microtubule assembly, the regulation and mechanics of spindle assembly, and the generation of cellular and embryonic axes. However, little information has been available regarding the organization of cytoskeletal elements in vivo during oogenesis. In large part, this results from the unique challenges posed by immunofluorescence microscopy of these large cells (up to 2 mm in diameter). Preservation and staining of labile cytoskeletal elements is often difficult in large cells, due to the limited penetration of fixative and antibodies or other dyes. In addition, the accumulation of yolk during oogenesis renders larger oocytes, eggs, and early embryos opaque. Finally, cell size also often limits the magnification and resolution possible, since the details of cytoskeletal organization in large oocytes and eggs often are obscured by fluorescence from outside of the focal plane when objectives with high numerical aperture are used.