Exposure of the ovaries of Triturus cristatus to actinomycin D at a concentration of 100 µg/ml causes characteristic changes in the peripheral nucleoli and other nuclear organelles in oocytes of 0.6-1.1 mm diameter. Viewed with the light microscope untreated oocytes contain nucleoli that stain uniformly with a variety of dyes. They also appear homogeneous tmder phase-contrast optics. After 2 or 4 h of in vivo incubation with actinomycin D, oocyte sections stained with Haidenhain's haematoxylin or viewed under phase-contrast optics show nucleoli composed of 2 regions. The more heavily stained or contrasted zone is crescent-shaped and directed away from the nuclear membrane. Neither sections stained with azure B bromide nor gallocyanin chrome alum show this feature. Ribonuclease digestion does not eliminate or alter it. Autoradiography with [3H]uridine indicates that all recently synthesized RNA is lost from the nucleolus during actinornycin D treatment. The zonation is not therefore a reflexion of RNA distribution. During recovery from actinomycin D poisoning there is a reduction in the degree of zonation shown by nucleoli which re-establish a normal appearance some 48 h after treatment.
Electron microscopy of peripheral nucleoli in oocytes sampled during this treatment indicates that the zonation is not associated with reorganization of ultrastructural components. During incubation with actinomycin D the coarse granules (20 nm diameter) are completely lost from the nucleolus. There is associated shrinkage of the nucleolus which after treatment is found to consist entirely of fibrils (5 nm thick) and small granules. The reappearance of the coarse granules during recovery is completed in about 48 h. It is thought that the loss of the granular component during treatment represents the movement of the 30-S precursor and the 18-s ribosomal unit from the nucleolus.
Some 20-30 µm inside the nucleus of untreated oocytes is a region containing many spheroidal bodies, less than 1.0 µm diameter. They have been termed micronucleoli and consist of granules 2.5-5 nm in diameter and fibrils of similar thickness. Actinomycin D treatment causes these components to segregate and eventually (within 24 h of treatment) the granular component is extruded. This component reappears during the second day after treatment. It is postulated that these micronucleoli represent the sites at which the 30-S ribosomal precursor undergoes its final maturation. The segregation of components induced by actinomycin D is probably the morphological manifestation of an abnormal metamorphosis of this precursor.
Treatment with actinomycin D also induces the immediate formation within the nucleus of crystalline bodies composed of lamellae 16 nm wide, 4 nm thick and with a centre-to-centre spacing of 8-10 nm. They are not present 24 h after treatment. They are thought to represent a protein fraction normally associated with periods of intense RNA synthesis.