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The Nucleolus and Meiosis During Microsporogenesis in Endymion Non-Scriptus (L.)

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The Nucleolus and Meiosis During Microsporogenesis in Endymion Non-Scriptus (L.) J. Cell Sci. 25, 111-123 (1977) 111 Printed in Great Britain THE NUCLEOLUS AND MEIOSIS DURING MICROSPOROGENESIS IN ENDYMION NON-SCRIPTUS (L.) B. T. LUCK AND E. G. JORDAN Biology Department, Queen Elizabeth College, University of London, Campden Hill Road, London WS 7 AH, England SUMMARY Stages of meiosis from the bluebell Endymion non-scriptus (L.) were studied by electron microscopy. The segregated components of the nucleolus at meiotic prophase underwent fragmentation and dissolution at pachytene-diplotene. Nucleoli were absent during both meiotic divisions and reformed on the nucleolus organizer into a fibrillar mass from scattered fibrillar components at the dyad and tetrad stages. It is argued that the fibrillar region shows continuity through nuclear division though undergoing structural transformations in the process. Nucleolar reformation occurs on condensed nucleolus organizers. Processing of the ribosomal precursors and the resumption of RNA synthesis is discussed in relation to the dispersal of the nucleolus organizer into the fibrillar region of the reformed nucleolus. INTRODUCTION Nucleolar ultrastructure during the mitotic cycle has been extensively studied by various investigators (Lafontaine & Chouinard, 1963; Jordan & Godward, 1969; Pickett-Heaps, 1970; Noel, Dewey, Abel & Thompson, 1971; Chouinard, 1966, 1971, 1975; Lafontaine, 1958; Lafontaine & Lord, 1974). It is generally agreed that the nucleolus suffers a general dissolution during late prophase of mitosis in higher plants; however, there are differing reports on the mode of its dissolution and the subsequent fate of the different parts (Lafontaine & Lord, 1969; Chouinard, 1971; Moreno Diaz De La Espina, Risueno, Fernandez-Gomez & Tandler, 1976). The origin of the material which constitutes the reappearing nucleoli is also still a matter of considerable uncertainty. Cytochemical studies of Allium cepa meristematic cells (Stockert, Fernandez-Gomez & Gimenez-Martin, 1970; Gimenez-Martin, De La Torre, Fernandez-Gomez & Gonzalez-Fernandez, 1974; Moreno Diaz De La Espina et al. 1976) support the earlier view that prenucleolar bodies, seen in the newly formed nucleus, arise from the so called pre-nucleolar material seen around the anaphase chromosomes (McClintock, 1934; Lafontaine, 1958). However, the contri- bution to the new nucleoli of the so called pre-nucleolar fibrillo-granular material found around the anaphase chromosomes is questioned by some workers (Swift, 1959; Chouinard, 1966; Lafontaine & Lord, 1974). The extent to which the new nucleolus is dependent on de novo synthesis has been followed in recent experiments employing inhibitors of RNA and protein synthesis (Gimenez-Martin et al. 1974; Semeshin, Sherudilo & Belyaeva, 1975; Risueno, Moreno Diaz De La Espina, Fernandez- Gomez & Gimenez-Martin, 1976). ii2 B.T. Luck and E. G. Jordan The synchrony of the meiotic process and the greater duration especially of the phase of nucleolar breakdown in the extended prophase of meiosis, together with the fact that there are 2 phases of reorganization, one after each division, only one of which leads to a normal interphase, makes it a useful and interesting system for studies on the nucleolar cycle. As previously reported (Jordan & Luck, 1976), concomitant with nucleolus segregation during meiotic prophase in Endymion non-scrtptus, the nucleolus organizer emerges to the surface of the nucleolus. In this paper we report the various rearrange- ments of the nucleolar zones in the prophase breakdown and the subsequent reorganiz- ation events after each of the 2 meiotic divisions. This work indicates that some of the constituents of the new nucleolus are synthesized before nucleolar reorganization. Thus the understanding of the nucleolus becomes not only the understanding of the synthesis of nucleolar materials but also their dispersal and reorganization at nuclear division. MATERIALS AND METHODS Endymion non-scriptus (L.) (Bluebell) plants were harvested in mid-January. The cyto- logical stage of the anthers in a floret was found from an aceto-orcein squash of one anther. The remaining 5 were sliced into 3% distilled glutaraldehyde in 01 M phosphate buffer, pH 6-8 at room temperature. The material was left in glutaraldehyde for 4-5 h, thoroughly rinsed in buffer, postfixed for 2-3 h at room temperature in 1% osmium tetroxide in the same buffer, dehydrated through an ethanol-propylene oxide series and embedded in Araldite. Sections were cut using a diamond knife, stained with aqueous lead citrate and post-stained with 10% (w/v) uranyl acetate in methanol (Stempack & Ward, 1964), and examined in an AEI EM 6B electron microscope. Labelling on figures a accessory nucleoli U lipid droplet c chromatin in mitochondrion cli chromosome nd nucleoid ci cytoplasmic imagination ne nuclear envelope f fibrillar region np nuclear pore ff fibrillar region fragment pb pre-nucleolar body g granular region psc polysynaptonemal complex if granular region fragment me remnant nuclear envelope go Golgi sc synaptonemal complex 1 lightly staining region V vacuole Figs. 1-3. Electron micrographs showing nucleolus segregation and fragmentation. Fig. 1. Segregated zygotene nucleolus with the nucleolus organizer in an external position. An accessory nucleolus is on the surface of the nucleolus organizer and cytoplasmic invaginations are present in the nucleus, x 12500. Fig. 2. Pachytenc-diplotene. High magnification of nucleolus organizing region showing fibrils of 5-125 nm in diameter. The accessory nucleoli have a similar fibril size to the fibrillar region: 5-10 nm, and the fibrils of the darkly staining chro- matin are 10-15 nm m diameter, x 32000. Fig. 3. Pachytene-diplotene. There is a clear separation of the various parts of the nucleolus; the fibrillar region has fragmented. An accessory nucleolus and a remaining synaptonemal complex are seen in the diffuse chromatin. Few nuclear pores are observed in the nuclear envelope, x 11000. The nucleolus during microsporogenesis 113 II4 B. T. Luck and E. G. Jordan The nucleolus during microsporogenesis 115 RESULTS Nucleolar segregation, separation and dissolution Zygotene nucleoli displayed the typical 'segregated nucleolus' appearance and also had some accessory nucleoli associated with the nucleolus organizer (Fig. 1). Accessory nucleoli had a similar staining intensity to the fibrillar region (Fig. 1), with fibrils between 5 and 10 nm in diameter. The chromatin fibrils were 10-15 nm m diameter (Fig. 2). By diplotene, the various components had separated (Fig. 3). These separate parts of the nucleolus also showed evidence of fragmentation (Figs. 3-6). During the final stages of nucleolar dissolution when the fibrillar and granular zones had moved apart from each other and were breaking up there was no indication of the fate of the granules or fibrils. The small areas of separate fibrillar and granular zones simply became smaller until they eventually disappeared. From the compactness of the fibrillar pieces during this process it seems that fibrils must be leaving the surface to reduce the size of the fragments, rather than gradually dispersing en masse by a general disaggregation. The granular region, however, although staying in recogniz- able pieces, does seem to disperse by becoming generally looser until the granules cannot be distinguished from the background. At this time there is an increase in the number of granules seen in the nucleoplasm. It is difficult to decide whether or not any of the nucleolar materials are contributing to the matrix of the recondensing chromosomes because they are still very dispersed at this time. During nucleolar dissolution large areas of the nuclear envelope remain intact (Figs. 3, 4). Frequently, large pieces of nucleolar granular zone were seen in apposition to the nuclear envelope (Fig. 6). Chromosomes at metaphase 1 and nucleolar reformation at the dyad stage Meiotic metaphase chromosomes showed a homogenous fibrous appearance (Fig. 7) with individual fibrils between 6 and 15 nm in diameter (Fig. 8). During chromosome decondensation at the time of the newly formed dyad, numerous fibrous bodies were seen adjacent to the chromosomes and free in the nucleoplasm (Fig. 9). As deconden- sation progressed these bodies decreased in number and increased in size (Figs. 10-12), and were seen to be composed of fibrils 5-10 nm in diameter (Fig. 12). These pre-nucleolar bodies coalesced on to the nucleolus organizer forming the main nucleolus which was composed of a fibrillar mass with a lightly staining zone on the surface (Figs. 13, 14), essentially similar to the inactive nucleoli described in Helianthus tuberosus (Jordan & Chapman, 1971). Figs. 4-6. Various aspects of nucleolar dissolution. Figs. 4, 5. Pachytene-diplotene, showing the fragmentation of the granular and fibrillar region of the nucleolus. Fig. 4, x 16500; Fig. 5, x 9500. Fig. 6. Diplotene. A large granular region fragment is seen in apposition to the remnant nuclear envelope, x 12750. n6 B. T. Luck and E. G. Jordan m The nucleohis during microsporagenesis 117 Chromosomes at anaphase 2 and nucleolar reformation at the tetrad stage Anaphase 2 chromosomes showed loosely arranged fibrils 6-15 nm in diameter (Fig. 15). Nucleolar reformation at the tetrad stage occurred in a similar fashion to that at the dyad stage, with the appearance of pre-nucleolar bodies which coalesced on to the nucleolus organizer (Fig. 16) to form the main nucleolus of the young tetrad, which was also composed of a large fibrillar mass with a lightly staining zone on
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