On the formation of amplified nucleoli during early Xenopus oogenesis

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Abstract

In Xenopus the genes for ribosomal RNA are selectively amplified during the early stages of oogenesis and give rise to over 1000 extrachromosomal nucleoli. These oocyte nucleoli are unique in that they contain very high copy numbers of rRNA genes, are not attached to chromosomes, and lack nonribosomal DNA. How the amplified rRNA genes induce the formation of multiple nucleoli is as yet poorly understood. To gain some more insight into this assembly process we have studied nucleolar development in early previtellogenic Xenopus oocytes. By using light and electron microscopic immunocytochemistry, in situ hybridization, and the TdT-method for detection of DNA, we have identified three separate structural entities which are related to the three components of the functionally active nucleoli present in midsized oocytes. Besides fibrillar and granular bodies we describe a novel rod-like structure which contains the pol I-specific transcription factor UBF. Notably, rDNA emerges from these rods forming a filamentous layer. These results reinforce UBF’s role as an architectural element involved in the spatial organization of the rRNA genes. We consider the rod-like rDNA/UBF complexes as extrachromosomal nucleolar organizers which are first juxtaposed to preformed fibrillar bodies before both elements gradually fuse into a coherent nucleolar structure.

Introduction

The genes coding for ribosomal RNA (rRNA) are selectively amplified during early amphibian oogenesis. As a consequence, a single Xenopus laevis oocyte nucleus contains about 30 pg of extrachromosomal rDNA, corresponding to roughly two million copies of rRNA genes. These amplified rRNA genes are arranged in the form of DNA circles with varying numbers of tandemly repeated gene units and located in over 1000 nucleoli per oocyte nucleus (for references see Gall, 1969; Macgregor, 1972; Miller, 1981; Thiebaud, 1979). In midsized oocytes the amplified rRNA genes are heavily transcribed, as revealed by the maximum packing density of polymerase molecules and their nascent transcripts along the gene axes in electron microscopic chromatin spreads (Martin et al., 1980; Scheer et al., 1977). This, in conjunction with the large copy number of the rRNA genes, allows the amphibian oocyte to support unusally high rates of ribosome production. While a rapidly growing somatic cell synthesizes 10–100 ribosomes per second, a single Xenopus oocyte produces approximately 300 000 ribosomes in the same time span (Scheer, 1973).

Recently, we have studied the functional organization of the amplified nucleoli in midsized Xenopus oocytes, i.e., at a stage of active ribosome biosynthesis (Mais and Scheer, 2001). Immunocytochemistry and mapping of transcription sites showed that the architecture of the oocyte nucleoli is remarkably similar to that of “standard” nucleoli of somatic cells. In particular, we have identified the fibrillar centers (FC) as the nucleolar subdomains which harbor the transcriptionally active rRNA genes and are selectively labeled with antibodies to RNA polymerase I (pol I) and the pol I-specific transcription factor UBF. The other major components, i.e., dense fibrillar component (DFC) and granular component (GC), were also unequivocally identified by immunogold EM using antibodies against appropriate marker proteins (fibrillarin and nucleolin; Mais and Scheer, 2001). Thus, we have the necessary tools at hand to analyze the formation of the amplified nucleoli during early oogenesis.

How a separate rDNA molecule is capable of organizing the assembly of a large (up to 15 μm in diameter) extrachromosomal nucleolus with its elaborate arrangement of its subcomponents is as yet poorly understood. In Xenopus, the rRNA genes are amplified very early in oogenesis at the zygotene/pachytene stage and first form an intranuclear caplike aggregate. At late pachytene, multiple small fibrillogranular bodies develop within this region (Coggins, 1973; Van Gansen and Schram, 1972). Later, in the early diplotene stage, the rDNA cap gradually disintegrates and the nucleolar bodies disperse throughout the nucleus. In Xenopus oocytes with diameters of about 50 μm (“diplotene B” according to Van Gansen and Schram, 1972) these nucleolar bodies have been described as bipartite structures composed of a silver-stainable fibrillar aggregate surrounded by caplike granular material (Boloukhere, 1984; Van Gansen and Schram, 1972; Williams et al., 1982). In somewhat larger oocytes (“diplotene C,” about 100 μm in diameter), the nucleolar bodies assume a ribbon-shaped configuration consisting of alternating fibrillar and granular bodies (Van Gansen and Schram, 1972). Throughout this early phase of oogenesis, Feulgen-positive granules are juxtaposed to the nucleolar structures, indicating that the rDNA is highly condensed and closely associated with, but not integrated into, the nucleolar elements (Spring et al., 1996; Thomas and Schram, 1977). Towards the end of diplotene C, the nucleolar ribbons start to fragment into multiple spherical nucleoli, concomitant with a gradual loss of the granular component, resulting in numerous purely fibrillar bodies in diplotene E stage (oocyte diameters 150–400 μm, Van Gansen and Schram, 1972).

In the present study, we have reexamined the assembly of the amplified nucleoli in young Xenopus oocytes by immunocytochemistry at the light and electron microscopic (EM) levels. Most importantly, we have identified the rDNA-containing structures and provide evidence that the transcription factor UBF is involved in the spatial organization of the amplified rDNA.

Section snippets

Biological materials and antibodies

Young X. laevis females at ages between 2 and 3 months after metamorphosis were anesthetized in MS222 (Serva Heidelberg, Germany); the ovaries were dissected and placed in modified Barth’s medium (Peng, 1991). Previtellogenic oocytes were staged according to Van Gansen and Schram (1972). In the present study, we examined oocytes from late diplotene B (approximately 70 μm in diameter) to early diplotene C (approximately 100 μm in diameter). These previtellogenic oocytes correspond to stage I

Morphology of the nucleolar precursor structures

To study the in situ formation of amplified nucleoli during early oogenesis, ovaries were dissected from freshly metamorphosed Xenopus females. When examined under the light microscope, the small transparent ovaries were seen to contain a more or less uniform population of previtellogenic stage I oocytes in the early diplotene stage of prophase I (Fig. 1). A few larger oocytes representing more advanced diplotene stages were interspersed between them and characterized by the presence of a

Discussion

Although ultrastructural features of the formation of multiple nucleoli during early Xenopus oogenesis have been studied in detail in earlier EM studies (Boloukhere, 1984; Coggins, 1973; Van Gansen and Schram, 1972, Van Gansen and Schram, 1974), the relationship of the newly developing nucleolar structures to the amplified rDNA remained obscure. After dispersal of the rDNA-containing cap structure in late pachytene/early diplotene stage oocytes, the fate of the amplified rDNA could no longer be

Acknowledgments

We thank M. Christensen for mAb P2G3, C. Dreyer for mAb P7-1A4-4, G. Reimer for mAb 72B9 and the autoimmune serum S18, and Silke Hofbauer for skillful technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (priority program “Functional Architecture of the Cell Nucleus,” Grant Sche 157/12-3).

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