J. Cell Set. 31, 79-100 (1978) Printed in Great Britain © Company of Biologists Limited 197S ULTRASTRUCTURE AND ACTIVITY OF THE NUCLEOLAR ORGANIZER IN THE MOUSE OOCYTE DURING MEIOTIC PROPHASE C. MIRRE AND A. STAHL, with the technical assistance of A. de Lanversin and C. Moretti (CNRS) Laboratoire d'Histologie et Embryologie II, Faculty de Midecine, 27, boulevard Jean-Moulin, 13385, Marseille, CEDEX 4 SUMMARY The mouse oocyte is the site of nucleolar synthesis during pachytene. The chromosomes containing a nucleolar organizer are attached to the nuclear envelope by their paracentromeric heterochromatin, either alone or by taking part in the formation of a chromocentre. The nucleolus appears at the junction of the paracentromeric heterochromatin with the euchromatic portion of the bivalent. In this zone, s-o-nm-diameter fibres, thinner than those of the rest of the chromosome (ico nm), extend from the lateral element of the synaptonemal complex up to the nucleolar fibrillar centre in which they penetrate. At the onset of its synthesis, the nucleolus only contains the fibrillar centre and an electron-dense fibrillar component in continuity with the latter. Growth of the nucleolus often takes place in the form of a strand whose proximal end, in contact with the fibrillar centre, is formed by preribosomal fibrils and whose distal end is at first nbrillo-granular then granular. Following brief incorporation of tritiated uridine, nucleolar labelling is active in oogonia. No ribosomal RNA-synthetic activity is revealed during leptotene and zygotene. Incorporation resumes at mid-pachytene, with labelling located over the electron-dense fibrillar component adjacent to the fibrillar centre. These observations suggest that the rDNA is located in both the fibrillar centre and its associated electron-dense fibrillar component and that the rDNA transcription occurs in the latter. INTRODUCTION The nucleolar organizer has been localized in the secondary constriction of certain metaphase chromosomes (Heitz, 1931; Heitz & Bauer, 1933; Hsu, Brinkley & Arrighi, 1967; Smetana&Busch, 1970). Localization of the ribosomaJ cistrons in the secondary constriction has been confirmed by in situ hybridization in Xenopus laevis and Xenopus mulleri (Pardue, 1974), man (Henderson, Warburton & Atwood, 1972; Evans et al. 1974), the gibbon Hylobates lar (Warburton, Henderson & Atwood, 1975), the kangaroo rat (Hsu et al. 1975), the Indian muntjac (Pardue & Hsu, 1975) and the mouse (Henderson, Eicher, Yu & Atwood, 1974; Elsevier & Ruddle, 1975; Henderson, Eicher, Yu & Atwood, 1976). In situ hybridization also reveals that the ribosomal cistrons may be detected in chromosomal regions not displaying secondary constrictions (Hsu, Spirito & Pardue, 1975; Elsevier & Ruddle, 1975; Henderson et al. 1976). In the interphase nucleus identifying the site of the nucleolar organizers is more 6-2 80 C. Mirre and A. Stahl difficult to achieve. Chouinard (1971) and Goessens (1973, 1974, 1976) were the first to suggest that the fibrillar centre of the nucleolus described by Recher, White- scarver & Briggs (1969), corresponds to the nucleolar organizer. Nevertheless, the relations between the fibrillar centre and a specific region of the chromosomes are impossible to demonstrate in the interphase nucleus. These relations can only be studied in prophase nuclei where the chromosomes are both individualized and accessible to analysis as is the case during meiosis. This situation has been exploited for studying the nucleolar organizer in plants (Braselton & Bowen, 1971; Gillies, 1973; Esponda & Gimenez-Martin, 1975; La Cour & Wells, 1975). Jordan & Luck (1976) clearly showed that in the bluebell Endymion non-scriptus the nucleolar organizer corresponds to the fibrillar centre. During meiotic prophase in the quail, the chromosomes containing the nucleolar organizer present highly characteristic relations with the fibrillar centre of the nucleolus. Chromatin fibres emanating from these chromosomes penetrate into the fibrillar centre (Mirre & Stahl, 1976). In the quail oocyte, localization of the ribosomal cistrons in the fibrillar centre has been confirmed by in situ hybridization (Knibiehler, Navarro, Mirre & Stahl, 1977). In the microsporocytes of those plants which have been studied, the peripheral location of the fibrillar centre (or its equivalent) with respect to the nucleolus is due to nucleolar segregation related to the arrest of nucleolar activity (Jordan & Luck, 1976). An analogous phenomenon of segregation, which parallels decreased incorpora- tion of tritiated uridine, has been observed in the human spermatocyte during pachytene stage (Tres, 1975). This is not the case in the quail oocyte, where a peripherally situated fibrillar centre is observed during a phase of synthesis of the nucleolar components (Mirre & Stahl, 1976). Nevertheless, on purely morphological grounds, it is highly difficult to distinguish between the segregation resulting from inactivation, identical to that caused by drugs which inhibit ribosomal RNA synthesis, and the separation of the fibrillar and granular components resulting from progressive synthesis and maturation originating in a DNA transcription centre. In this study, the relations between the secondary constriction of the chromosomes containing a nucleolar organizer and the fibrillar centre of the nucleolus were analysed at the same time as ribosomal cistrons activity was studied by incorporation of tritiated uridine. The cell stages investigated extended from the oogonium up to the early diplotene oocyte. Fig. 1. Oogonium from a 15-day-old mouse embryo. Nucleolus displays a reticulated type of structure containing several fibrillar centres (arrowheads) surrounded by electron-opaque fibrils and separated from the nuclear envelope by a heterochromatic mass, x 9000. Fig. z. Preleptotene chromosomal condensation stage in the nucleus of a 15-day-old oocyte. x 7000. Nucleolus contains fibrillar centres (inset, arrows), x 14000. Fig. 3. Leptotene oocyte in a 16-day-old mouse embryo. Axial cores are visible (arrows). Nucleoli exhibit a granular texture and fibrillar centres are absent, x 9600. Inset: detail of enlarged nucleolus. x 19000. Fig. 4. Early pachytene in a 20-day-old mouse embryo. Several bivalents are associated by their centromeric heterochromatin, constituting a chromocentre in contact with the nuclear envelope: no newly synthesized nucleolus is observed, x 13750. Niicleolar organizer in mouse meiotic oocyte 81 TPWK- v# "^t* r :?(i™'lft. TF^X 82 C. Mitre arid A. Stahl MATERIALS AND METHODS Pregnant SWISS OF i mice were used. The ovaries of 2 embryos from each mouse were taken ranging from the 13th day of pregnancy to birth and from 1 to 3 days post partum. Specimens destined for morphological study were treated by double fixation with 3 % glutaraldehyde in o-i M phosphate buffer (pH 7-2) containing o-i M sucrose, for 15 min at 4 °C. They were then washed rapidly in the buffer solution and postfixed in 2 % osmium tetroxide in identical buffer for 20 min at 4 °C. After dehydration in a graded series of acetone, the specimens were embedded in Epon. Sections of silver interference colour were obtained on a Reichert OMU2 ultramicrotome with a diamond knife. The sections were contrasted with uranyl acetate and lead citrate. For high resolution autoradiography, the entire ovaries were incubated 30 or 45 min at 37 °C in 1 ml of 80% Eagle and 20% calf serum medium containing 100/tCi/ml of [3H]- uridine ([3H]uridine, sp. act. 24 Ci/mM Lot no. 276 CEA France). After the above described fixation and before postfixation, the specimens were repeatedly washed during 30 min in the buffer in order to remove the unincorporated radioactive precursor. After dehydration and embedding in Epon, light-gold interference-coloured sections were realized for the following technique according to Granboulan (1965) and Salpeter & Bachmann (1972). The sections were transferred to a collodion-coated (2 % colloidine in iso-amylacetate) slide. The sections on the slide were stained with 5 % aqueous uranyl acetate solution, freshly prepared, in 95 % ethanol v/v (for 30 min in dark) rinsed off in 50 % ethanol and air-dried before staining with lead citrate for 2-5 min. The stains were rinsed off by flushing with distilled water. A thin carbon layer (about 5 nm) was vacuum evaporated over the stained sections. The slides were then coated with a diluted Ilford L4 emulsion by the dipping method: 1 vol. of Ilford L4 emulsion was melted in 4 vol. of 40 °C distilled water during 1 h and slowly stirred every 15 min. Dried slides were stored at 18 °C in sealed slide boxes with a dessicant (P,O5). After exposure for 4 months, the preparations were developed in Microdol-X (Kodak) for 4 min at 18 °C and rinsed off in distilled water. Fixation was carried out with Kodak Rapid Fixer for 3 min at 18 °C and the slides were then washed 3 times, 5 min each, in distilled water. The specimen was stripped off the glass on to a clean water surface and copper grids (200 mesh) were placed over the sections. The specimens on the grids were then picked up with a filter paper. After drying, the grids were carefully released from the remnant film. The collodion film was thinned for 2-5 min in iso-amylacetate. All the preparations were examined using a Siemens Elmiskop 101 microscope at 80 kV with an objective aperture of 50 /im. OBSERVATIONS The oogonial nucleus contains homogeneously distributed chromatin. No axial cores are visible. The large nucleolus displays a reticulated type of structure essentially Fig. 5. Late pachytene in a 20-day-old mouse embryo. Two newly synthesized nucleoli (arrowheads) are in contact with 2 bivalents, whose synaptonemal complexes are partly within the chrome-centre, x 5000. Fig. 6. Detail of preceding figure. The developing nucleolus exhibits a fibrillar texture and displays a fibrillar centre (/c) in continuity with the bivalent chromatin. x 43 000. Fig. 7. Late pachytene in a 20-day-old mouse embryo. The nucleolus develops in close relation to a bivalent whose centromeric heterochromatin is inserted on the nuclear envelope, x 13000. Fig. 8. Detail of preceding figure.