Absence of rDNA amplification in the uninucleolate oocyte of the cockroach Blattella germanica (Oorthoptera: Blattidae)

Amplification of the genes coding for ribosomal RNA oocurs in the oocytes of a wide variety of organisms. In oocytes of various species of crickets (Orthoptera: Gryllidae) the amplified DNA is contained in a large extrachromosomal DNA body. Multiple nucleoli form about the periphery of the DNA body during the diplotene stage of meiosis I. In contrast to the general pattern of orthopteran oocytes, oocytes of the cockroach Blattella germanica demonstrate a single large nucleolus instead of many nucleoli. In order to determine whether the genes coding for rRNA are amplified in the oocytes of B. germanica, the relative amount of rDNA in oocytes was compared with the rDNA content of spermatocytes and somatic cells. An extrachromosomal DNA body similar to that present in crickets is not present in B. germanica. A satellite DNA band which contains nucleotide sequences complementary to rRNA accounts for approximately 3-5% of the total DNA in somatic and in male and female gametogenic tissues. Female cells contain approximately twice as much rDNA as do male cells. An XX-XO sex-determining mechanism is operative in B. germanica. In situ hybridization with rRNA indicates that the nucleolar organizer is located on one end of the X chromosome and that oocytes do not contain more than twice the amount of rDNA found in spermato cytes. The data indicate that rDNA is not amplified in the uninucleolate oocyte of B germanica.     

Nucleolar DNA in oocytes of crickets: representatives of the subfamilies Oecanthinae and Gryllotalpinae (Orthoptera: Gryllidae)

A large extrachromosomal mass of Feulgen positive material, the DNA body, has been visualized in early prophase oocytes of crickets (Orthoptera: Gryllidae) representative of the closely related subfamilies Gryllinae and Nemobiinae. A similar structure is present in oocytes of representatives of two subfamilies of crickets (subfamilies Oecanthinae and Gryllotalpinae) which taxonomically and phylogenetically are quite separate from those mentioned previously. In situ hybridization demonstrates that the body contains amplified copies of genes coding for ribosomal RNA. Unlike the DNA body in early diplotene oocytes of representatives of the subfamily Gryllinae, which is closely associated with the developing nucleolar apparatus, the DNA body in oocytes of the Oecanthinae and Gryllotalpinae cannot be demonstrated during diplotene. In the Oecanthinae, the nucleolar apparatus of early diplotene stage oocytes is composed of four to seven separate structures, the ribonucleoprotein of which has a characteristically lamellated appearance. During late diplotene, these nucleoli give rise to many smaller structures which are distributed throughout the germinal vesicle. In early diplotene stage oocytes of Scapteriscus acletus (Subfamily: Gryllotalpinae), the nucleolar apparatus consists of a single compact mass of ribonucleoprotein. In contrast to the oocytes of all other crickets that have been studied, the nucleolus of S. acletus remains single throughout diplotene. In situ hybridization analysis indicates that the amplified genes coding for rRNA which are localized in the DNA body of early prophase oocytes become incorporated into this compact nucleolar mass. Differences in nucleolar structure appear to reflect differences in the organization of amplified genes coding for rRNA.     

Amplification of ribosomal genes and formation of extrachromosomal nucleoli in oocytes of starfish Henricia hayashi (Asteroidea: Echinasteridae)

DNA and RNA specific dyes, Ag-NOR staining and in situ hybridization were used for studying the nucleolar apparatus in the growing oocytes of Henricia hayashi (Asteroidea: Echinasteridae). A plasmid containing ribosomal genes of Drosophila melanogaster (Kolchinsky et al., 1980) labelled with 3H by nick-translation served as an rDNA probe. Multiple extrachromosomal nucleoli are formed by the cascade type as a result of growth and subsequent fragmentation of the chromosomal (primary) rDNA body and its derivative extrachromosomal (secondary) rDNA bodies. Ribosomal genes were shown in all nucleolar structures. Argentophilia of the primary and secondary DNA bodies appears to be due to the dense packing of the rDNA-containing material. Ag(+) NORs were detected in the extrachromosomal multiple nucleoli and NOR complexes. Amplification of rDNA is a highly probable conclusion from the existing data.     

Ultrastructure of trophocyte nuclei in polytrophic ovarioles of Chrysopa perla (Neuroptera)]

The light and electron microscope and autoradiographic studies (H3-uridin incorporation) were carried out on the trophocyte nuclei of imago polytrophic ovarioles of Chrysopa perla (Neuroptera), from the trophocyte differentiation up to their degeneration. Like the oocytes, one of the seven nurse cells o every ovariole chamber contains extrachromosomal DNA bodies. This nurse cell is formed during differential mitoses in the germarium as one of two prooocytes. In contrast to extrachromosomal DNA of oocytes the trophocyte DNA bodies are less active structures. Several (2--4) complex nucleoli develop in the trophocytes of Chrysopa in the early stages of oogenesis. They consist of three main components: the chromatin mass, fibrillar bodies and granular strands. Such nucleoli grow, through increasing in number of fibrillar bodies and granular strands. They are most developed by the start of the vitellogenesis. At the middle vitellogenesis the general nucleolar structure modify due to the beginning of trophocyte degeneration. The consecutive stages of nuclear degeneration are described. The trophocyte nucleoli synthesize RNA still in germarium. The most intensive RNA synthesis is observed at the beginning of the vitellogenesis to decrease by the beginning of trophocyte degeneration.     

Fate of amplified nucleoli in Xenopus laevis embryos

We have followed the fate of two components of extrachromosomal nucleoli, amplified ribosomal DNA (rDNA) and 7.5 kb precursor rRNA, during early embryogenesis of Xenopus laevis. Other workers have shown that the amount of amplified rDNA accumulated during oogenesis remains unchanged through the 16-cell stage of embryogenesis. Here we show that as embryonic cleavage continues, the amount of amplified rDNA decreases until it is no longer detectable in the early gastrula embryo. In contrast, the amount of 7.5 kb precursor rRNA in eggs, early cleavage stage embryos, or blastula stage embryos is the same as in oocyte nuclei. Since no rRNA synthesis occurs during these early stages, we conclude that the precursor rRNA sequences synthesized in the oocyte are neither processed nor degraded during early development. The amplified rDNA is not replicated in the early embryo even though the chromosomal DNA of the embryo replicates every 30 min during the first 7.5 hr of embryogenesis. When amplified rDNA is purified and then injected into cleaving embryos, however, we find that it is replicated. This finding suggests that some factor(s) prevents the endogenous amplified rDNA from responding to the cellular replication signals. We show that methylation of cytosine in the rDNA is not related to the DNA's capacity for replication in this system since amplified (unmethylated) and chromosomal (methylated) rDNA are both replicated when injected into embryos. The methylation pattern of these rDNAs appears to be maintained after replication in the embryo.     

“Micronucleoli” in theXenopus germinal vesicle

The germinal vesicle of the Xenopus oocyte contains 1500 or more extrachromosomal nucleoli that are assembled on amplified copies of the rRNA genes. Many of these nucleoli have diameters of 10–15 μm, but some are much smaller, ranging down to 1 μm or less. Morphologically the smaller nucleoli or ”micronucleoli” resemble the similarly sized B snurposomes, but they can be recognized with appropriate antibody probes (e.g., anti-nucleolin and anti-fibrillarin). We describe here a sensitive fluorescent staining technique that uses avidin and propidium iodide to visualize the rDNA in the amplified nucleoli. Many large nucleoli stain about as brightly as haploid yeast nuclei on the same slides. They presumably contain about 12 Mb of DNA, equivalent to 900 rDNA repeats. The smallest micronucleoli display only a tiny dot of stain, which must correspond to relatively few rDNA repeats. Received: 8 January 1997; in revised form: 20 January 1997 / Accepted: 27 January 1997     

The relationship of ribosomal RNA synthesis to the formation of segregated nucleoli and nucleolus-like bodies

The relationship of ribosomal RNA (rRNA) synthesis to nucleolar ultrastructure was studied in partial nucleolar mutants of Xenopus laevis. These mutations are the result of a partial deletion of rRNA genes and therefore alow studies on nucleolar structure and function without using drugs that inhibit rRNA synthesis. Ultrastructural studies demonstrated that normal embryos have reticulated nucleoli that are composed of a loose meshwork of granules and fibrils and a typical nucleolonema. In contrast, partial nucleolar mutants in which rRNA synthesis is reduced to less than 50% of the normal rate have compact nucleoli and nucleolus-like bodies. The compace nucleoli contain granules and fibrils, but they are segregated into distinct regions, and a nucleolonema is never seen. Since other species of RNA are synthesized normally by partial nucleolar mutants, these results demonstrate that nucleolar segragation is related specifically to a reduction in rRNA synthesis. The nucleolus-like bodies are composed mainly of fibrils,and the number of such bodies are composed mainly of fibrils, and the number of such bodies present in the different nucleolar mutants is inversely related to the relative rate of rRNA synthesis. Although the partial nucleolar organizers produce segregated nucleoli in these mutants, they organize morphologically normal, but smaller, nucleoli in heterozygous embryos. Alternative explanations to account for these results are discussed.     

Structure of the germinal vesicle during oogenesis in leech Glossiphonia heteroclita (Annelida, Hirudinea, Rhynchobdellida)

Oogenesis in the glossiphoniid leech Glossiphonia heteroclita (Hirudinea, Rhynchobdellida) is nutrimental, i.e., the growing oocyte is supported by specialized germline cells, the nurse cells. The main function of the nurse cells is to provide oocytes with cell organelles and RNAs (mainly rRNA). However, in studied leech species, irrespective of the nutrimental mode of oogenesis, the germinal vesicle (GV = oocyte nucleus) seems to be very active in rRNA production. As shown in the present study, during early previtellogenesis in the GV the meiotic chromosomes and prominent primary nucleoli occur. In late previtellogenesis the chromosomes condense and occupy a limited space of nucleoplasm in close vicinity to primary nucleolus, forming a karyosome. At the onset of vitellogenesis several prominent extrachromosomal DNA bodies appear in close association with the karyosome. At the same time, the primary nucleolus is no longer visible in the GV. As vitellogenesis proceeds the extrachromosomal DNA bodies undergo fragmentation and numerous spherical, RNA- and AgNOR-positive inclusions occur in the nucleoplasm. They are regarded as multiple nucleoli. Finally, in late oogenesis numerous accessory nuclei are formed in close proximity to the nuclear envelope. They usually contain one dense body, morphologically similar to multiple nucleoli. The amplification of rDNA genes, the occurrence of extrachromosomal DNA bodies, as well as the presence of multiple nucleoli and accessory nuclei are described for the first time in the phylum Annelida.