THE NUCLEOLUS AND ITS GENES IN AMPHIBIAN OOGENESIS
Authors: MACGREGOR HERBERT C. 
Published: January 2008

Summary1. Amphibian oocyte nucleoli may be solid and round, or they may take the form of beaded rings. Transformation of the nucleoli from the solid form to the ring form and back again may be a regular feature of oogenesis. The variation in circumference of freshly isolated unstretched ring nucleoli is continuous over a range of about fivefold. The longest rings may measure several hundred micra. All oocyte nucleoli have two structural zones, core and cortex. The core consists of finely fibrous material and is analogous to the ‘pars amorpha’ of earlier authors. The cortex has a granular fine structure and is analogous to the ‘nucleolonema’. The beads of ring nucleoli have both core and cortex components. Some solid nucleoli are multicored.2. When all nucleoli are single cored, the number of nucleoli in an oocyte nucleus remains constant throughout the lampbrush phase of oogenesis. When nucleoli are multicored, the number of nucleoli may vary, but the number of nucleolar cores remains constant.3. Oocyte nucleoli of between 6 and 12μ diameter have dry masses of between 50 and 200 μμg. 610% of the nucleolar dry mass is attributable to RNA.4. Each nucleolus has a small DNA component. This component is in the form of a long circular duplex. In solid nucleoli it meanders through the substance of the nucleolar core. It forms the axis of a ring nucleolus and the structural integrity of the ring is maintained by the DNA component.5. Both round and ring types of nucleoli from some salamanders and frogs have a membrane component attached to them. This takes the form of a membranous tube or a chain of vesicles ending near to or within a depression in the surface of the nucleolar core. Nothing is known at present of the function of the nucleolar membrane.6. Nucleoli are sites of synthesis of ribosomal RNA. The DNA of the nucleolar core has the base sequence needed for transcription of ribosomal RNA. RNA is transcribed, complexed with protein and eventually distributed between 50S and 30S ribonucleoprotein particles. The 30S particle does not accumulate in the nucleolus. The 50S particle matures in the nucleolus and probably constitutes a substantial portion of the nucleolar cortex.7. During the pachytene stage in ovaries of young amphibia 20–30 μμg. of extra‐chromosomal nucleolar DNA are synthesized. In early diplotene this DNA disperses into thousands of tiny granules which become distributed over the inner surface of the nuclear envelope. A nucleolus then grows in association with each granule.8. Isolation and purification of nucleolar DNA from pachytene oocytes, and from germinal vesicles, has shown that it contains the sequences for 18 and 28S ribosomal RNA, and it represents the products of an amplification of the genes for ribosomal RNA.9. In Xenopus the gene complex which is amplified consists of about 450 tandem repeats of a region of double stranded DNA which includes sequences complementary to 28 S and 18 S ribosomal RNA alternating with a spacer sequence. The density of the ribosomal DNA, including the spacer DNA, in somatic cells of X. laevis is 1.7236 g. cm‐3, and that of the oocyte amplified rDNA is 1.729. Both DNAs are 67% G + C. The somatic rDNA has 4.5% 5‐methyl‐deoxycytidylic acid which accounts for its lower density. The spacer sequences are uniform within a species, but differ from one species to another.10. In oocytes, rDNA is amplified by using one or both of the chromosomal nucleolar organizers as a primary template. Amplification starts in oogonia and proceeds slowly until the pachytene stage. Rapid synthesis of DNA takes place in pachytene and early diplotene stages over a period of about 20 days. It seems likely that DNA synthesis proceeds in a cascade pattern such that each new replica itself serves as a template for further replication. Evidence for synthesis of a primary RNA template upon which the amplified rDNA is made is equivocal.11. Whatever the size of the nucleolar organizer in an amphibian, and however many ribosomal cistrons it may incorporate, the amplification system in the oocyte is scaled accordingly, so that only a certain number of additional ribosomal cistrons and associated spacer are synthesized. Each of the eight germinal vesicles in an Ascuphus oocyte amplifies its rDNA to only one‐eighth of the extent seen in the single oocyte nuclei from other anurans. Mononucleolate Xenopus, whose oocytes have only half the usual complement of chromosomal ribosomal DNA, nevertheless amplify to the level found in the normal wild‐type individuals. Triploid Ambystoma, with hexaploid oocytes, appear to amplify their rDNA to three times the extent seen in their diploid relatives. It is clear that the level of gene amplification is related to the number of chromosomal organizers in the oocyte nucleus. It also seems that the level of amplification may be related to the volume of the oocyte nucleus.12. The lampbrush chromosomes of at least some amphibia carry attached oocyte nucleoli at sites whose positions correspond to those of the somatic nucleolar organizers. The lampbrush nucleolar organizer in the axolotl is often marked by round or ring‐like objects attached to the chromosome.13. These lampbrush nucleolar organizers may carry solid or ring‐like nucleoli. The attached nucleoli are thought to be permanently located there. They are not sequentially grown and detached from the chromosome. Attached ring or loop‐like nucleoli may form ‘double bridges’, indicating that at least in some cases they are an integral part of the chromosome axis. In Plethodon the lampbrush nucleolar organizer consists of a long stretch of non‐chromomeric axis that may appear single or double. There are ring or solid nucleoli attached at intervals along this axis. It is possible that these attached rings are products of amplification of the nucleolar organizer and that they became re‐attached to the chromosomal organizer by a process analogous to recombination during or soon after the main phase of amplification.