Ultrastructure and Activity of the Nucleolar Organizer in the Mouse Oocyte During Meiotic Prophase

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 incorporation 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

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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 fibrillarcentr e (/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. Chromatin fibres with a diameter clearly smaller than that of the other chromosome fibres penetrate into the fibrillar centre (/c). A zone of highly electron-opaque fibrils can be seen on either side of fibrillar centre (arrows). The remaining part of the nucleolus exhibits a fibrillar texture, x 85 000. Nucleolar organizer in mouse meiotic oocyte A 'M,:,^mm >/

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8 84 C. Mine and A. Stahl consisting of fibrillogranular components. Small fibrillar centres surrounded by electron-opaque fibrils can be seen. The nucleolus is either centrally or peripherally situated. In the latter case, the nucleolus is generally separated from the nuclear envelope by a mass of heterochromatin (Fig. i). At the stage of preleptotene condensation, the oocyte is characterized by increased density of the chromatin in the form of irregular blocks. Light-microscope studies during this stage show that these blocks, numbering 40 in the mouse, each correspond to a highly condensed chromosome (Hartung & Stahl, 1977). The nucleolus appears identical to the preceding stage (Fig. 2). At leptotene, the chromosomes can be seen as previously described by Solari (1969) in the mouse spermatocyte. In the nucleus, single axial cores are observed with chromatin fibrils inserted on them in a grossly radial distribution. At this stage, the appearance of the nucleolus has become modified. It is frequently less voluminous than in the oogonium and above all, presents an essentially granular texture (Fig. 3). No fibrillar centre can be observed. This same aspect is seen again at zygotene stage. At the onset of pachytene, the formation of chromocentres in contact with the nuclear envelope takes place. These chromocentres result from association of the centromeric heterochromatin from several bivalents (Fig. 4). We were able to count up to 7 bivalents, identifiable by their synaptonemal complex, associated in this manner (Fig. 5). The euchromatic portion of these bivalents extends more or less far within the nucleus, and this portion is only visible on favourable sections. These observations are identical to those reported by Hsu, Cooper, Mace & Brinkley (1971) in the spermatocyte of the mouse. At mid-pachytene stage, small nucleoli appear in contact with certain bivalents whose heterochromatin constitutes part of the chromocentre (Figs. 5-8). The nucleolus may also be synthesized in contact with an isolated bivalent (Fig. 9). The extremity of this bivalent is attached to the nuclear envelope. A synaptonemal complex is present in the axial region of the bivalent. Heterochromatin composed of thick and densely packed chromatin fibres surrounds the synaptonemal complex for about 1-5 /mi. The newly formed nucleolus always appears beyond this heterochro-

Fig. 9. Formation of a band-shaped nucleolus in the nucleolar-organizer region of a bivalent. The latter is attached on the nuclear envelope by its centromeric heterochromatin. The nucleolus successively displays: a fibrillar centre (very small on this section, short arrow); a narrow dense fibrillar zone (long arrow); a long purely fibrillar segment (/); a fibrillo-granular segment (fg) and a granular zone (g). This arrangement shows that preribosomal RNA is synthesized as fibrils which progressively mature into granules, x 54000. Fig. 10. Genesis of 2 nucleoli, each of which is in relation to the nucleolar-organizer region belonging to one chromosome of the bivalent. Beginning at the nuclear envelope (ne) the following structures are observed: the wedge-shaped zone of centromeric heterochromatin (he) composed of coarse fibres; the secondary constriction composed of approximately 50-nm-diameter fibres (circle) which penetrate into the fibrillar centre (/c) of one of the nucleoli; part of the euchromatin (ec) constituted by fibres of about 100 nm diameter. Note that one of the nucleoli is only composed of a fibrillar centre partially surrounded by an electron-dense fibrillar zone (/~). x 70000. % 86 C. Mine and A. Stahl matic zone at precisely the beginning of the euchromatic segment. The nucleolar organizer corresponds to the chromosomal region which forms the nucleolus. Quite frequently, simultaneous and approximately symmetrical formation of 2 nucleoli, on either side of the synaptonemal complex, is observed in the region of the nucleolar organizer (Figs. 10-14). This observation suggests that the 2 homologous nucleolar organizers, present together in the same bivalent, underwent activation at the same moment. Synchronous transcription of the ribosomal cistrons in 2 paired chromosomes yields 2 nucleoli, which are first separated at their points of origin and may sometimes fuse together at their essentially granular, distal regions (Fig. 13). The relations between the chromosome containing the nucleolar organizer and the nucleolus are highly characteristic. Facing the nucleolus, the chromosome presents chromatin fibres of about 5-0 nm diameter. These fibres are located in a segment which never exceeds 0-25 /tm in length. Beyond this segment, the diameter of the euchromatic fibres is approximately io-o nm. The 5-o-nm fibres extend from the lateral element of the synaptonemal complex up to the nucleolus. The latter always displays a fibrillar centre oriented toward the chromosome. The 5-o-nm fibres penetrate into the fibrillar centre where they are no longer visible. This occurs since the 5-o-nm fibres cannot be distinguished from the network of fibrils belonging to the fibrillar centre which display moderate electron opacity (Figs. 6, 8, 10—12, 14). The fibrillar centre is largely surrounded by fibrils of high electron opacity (Figs. 8, 10-13). As the nucleolus progressively grows in size the fibrils extend out to form, one or several irregular bands (Figs. 7-9, 11, 13). In nucleoli at a more advanced stage of synthesis, a gradient distribution of the nucleolar components is observed. Beginning at the chromosomal region presenting the nucleolar organizer, the following structures can be seen: (1) the fibrillar centre; (2) a narrow and electron-opaque fibrillar zone; (3) a band-like zone of fibrillar texture; (4) a fibrillo-granular zone whose fibrils are progressively replaced by granules; and (5) a granular zone (Figs. 9, 11-13). It is obvious that the granular zone, composed of material at a more advanced stage of maturation, underwent synthesis prior to that of the fibrils, which are still in contact with the fibrillar centre. Morphological analysis alone already suggests that synthesis of the nucleolar components occurs in close relation to the fibrillar centre. At the onset of diplotene, the chromosomes begin to unravel and the synaptonemal complex to disappear. The disjoined chromosomes only display single axial cores.

Figs, ii, 12. Simultaneous and symmetrical synthesis of 2 nucleoli in relation to the 2 nucleolar organizers which are joined at the bivalent secondary constriction. Fig. 11. The bivalent is inserted on the nuclear envelope (ne) by its centromeric heterochromatin (lie). The secondary constriction (arrows) originates at the limit of this wedge-shaped zone and is composed of approximately 5-o-nm-diameter fibres which penetrate into the fibrillar centre of each nucleolus (fc). Beyond the fibrillar centre the upper nucleolus exhibits a fibrillar (/), fibrillo-granular (fg) and granular (g) zone, x 42000. Fig. 12. Note the fine fibrillar texture of the secondary constriction (sc) whose fibrils penetrate into the fibrillar centre (fc) of one nucleolus. The other nucleolus shows a conspicuous fibrillo-granular gradient, x 45 000. Nucleolar organizer in mouse meiotic oocyte 88 C. Mine and A. Stahl The nucleoli increase in size and progressively take on a reticular appearance. Occasionally and only temporarily, characteristic relations may be seen between the chromatin fibres of the nucleolar organizer and the fibrillar centre situated at the nucleolar periphery (Fig. 15). However, as diplotene progresses, these relations rapidly disappear. In the 1-day-old mouse oocyte, large reticular nucleoli can be seen displaying those components described by Chouinard (1971): (1) a fibrillo- granular component which constitutes the main part of the nucleolar reticulum; (2) several fibrillar centres which are smaller than at pachytene; and (3) electron- opaque fibrils distributed around each fibrillar centre. The interstices of the nucleolar framework contain a material analogous to that of the nucleoplasm. A mass of heterochromatin whose texture appears less dense than at pachytene, generally separates the nucleolus from the nuclear envelope.

Autoradiography Oogonia. Following incubation for 30 min, the nucleoli actively incorporate tritiated uridine (Fig. 17). The silver grains are essentially located over those regions where the electron-dense fibrillar component predominates. No labelling grains can be seen over the fibrillar centre per se, but are detectable over the surrounding electron-dense fibrils. In addition, silver grains are found over the euchromatic regions of the nucleus. Condensation stage. Active nucleolar incorporation of pHJuridine is observed, as in the previous stage. Labelling over the nucleolus is slightly reduced in comparison to that of the oogonium. Leptotene stage. The absence of nucleolar labelling is in contrast to the presence of silver grains lying over the nucleoplasm (Fig. 18). Zygotene stage. As during leptotene, the nucleolus displays no labelling, while silver grains can be seen overlying the chromosomes (Fig. 19). Pachytene stage. Nucleolar incorporation of tritiated uridine actively resumes at mid-pachytene. Following incubation for 30 min, labelling grains are located over the dense fibrillar component adjacent to the fibrillar centre (Figs. 20, 21). No silver grains are seen over the fibrillar centre, and the granular component is unlabelled. Labelling grains are fairly numerous throughout the rest of the nucleus. Such labelling is essentially located over the chromatin fibres which extend from either side of the synaptonemal complex.

Fig. 13. Simultaneous synthesis of 2 nucleoli which are fused together at their distal granular region (g) at late pachytene in a 20-day-old mouse embryo, x 40000. Fig. 14. Detail of preceding figure showing the relations between fibrils of the nucleolar organizer and the fibrillar centre (/c). x 116000. Fig. 15. Early diplotene in a mouse oocyte at postnatal day 1. The nucleolus shows a reticulated texture. One of the fibrillar centres (arrows) is still in connexion with a chromosome whose axial core (ac) is visible, x 19000. Fig. 16. Diplotene mouse oocyte at postnatal day 1. Several reticulated nucleoli are separated from the nuclear envelope by a heterochromatic mass: nucleoli contain several fibrillar centres (arrows) always surrounded by a dense fibrillar zone, x 10800. Nucleolar organizer in mouse meiotic oocyte

; * ^ 90 C. Mirre and A. Stahl When incubation is prolonged to 45 min, the labelling extends to the granular component of the nucleolus at late pachytene (Fig. 22). Early diplotene stage. Nucleolar incorporation of [3H]uridine actively occurs. Labelling grains are essentially seen over the dense fibrillar component. In the remaining regions of the nucleus, the silver grains are numerous, and are generally located over the chromatin fibres surrounding the single axial cores. During the different stages investigated, nucleolar and chromosomal grain counts indicated that a resumption in ribosomal RNA synthesis does indeed occur during pachytene (Table 1).

DISCUSSION Our observations demonstrate that synthesis of one or several nucleoli takes place at late pachytene in close connexion to the secondary constriction region of certain bivalents. In this region, 5-o-nm-diameter fibres extend from the lateral element of the synaptonemal complex up to the fibrillar centre of the forming nucleolus, and penetrate into the latter. The newly synthesized nucleolus displays a gradient distribution of its components. From the fibrillar centre to the opposite extremity of the nucleolus, the latter is fibrillar, then fibrillo-granular and finally granular. This observation alone suggests that the nucleolar components are synthesized in the region of the fibrillar centre since synthesis of the fibrils precedes that of the granules (Granboulan& Granboulan, 1965; Bernhard, 1966; Noel, Dewey, Abel & Thompson, 1971; Recher, Briggs & Parry, 1971; Smetana & Busch, 1974). The relations observed between the nucleolar chromosome and fibrillar centre in the mouse oocyte are largely similar to those we described in the quail oocyte. In the latter case as well, chromatin fibres emanating from a microchromosome containing the nucleolar organizer, penetrate into the fibrillar centre of the developing nucleolus (Mirre & Stahl, 1976). In the quail oocyte, comparison of results from in situ hybridization with ultrastructural data clearly indicates that the ribosomal cistrons are located in the fibrillar centre and probably in the surrounding bands of electron-dense fibrils as well (Knibiehler et al. 1977). In the mouse oocyte, penetration of the chromosomal fibres into the fibrillar centre is only interpretable by admitting that the latter is that region where the rDNA

Fig. 17. Oogonium of a 15-day-old mouse embryo. [3H]uridine incorporation, labelling time 30 min. Several grains are seen overlying the fibrillar strands. The fibrillar centre (arrow) is unlabelled. x 11700. Fig. 18. 15-day-old mouse embryo oocyte at leptotene. ['HJuridine incorporation, labelling time 30 min. The mostly granular nucleolus is unlabelled. Note the presence of silver grains overlying the chromatin. ac, axial core, x 33500. Fig. 19. Zygotene oocyte in a 15-day-old mouse embryo. Autoradiography as in preceding figures. The unlabelled nucleolus is composed only of granules. Silver grains are seen over the chromatin. x 21000. Nucleolar organizer in mouse meiotic oocyte w

6 92 C. Mine and A. Stahl fibres unravel to yield a spatial configuration which favours their transcription. These fibres probably exist as more or less contorted loops. Baker & Franchi (1967) have already reported that the fibres which are visible in the chromosomes of the human oocyte at diplotene form loops. In the mouse oocyte at pachytene, the rDNA chromatin fibres probably each form a loop of which at least a certain segment is entirely unravelled at the periphery of the fibrillar centre (Fig. 24). Using a whole mount technique, Kierszenbaum & Tres (1974^) showed that the mammalian bivalents display a lampbrush configuration determined by looping chromatin fibres. Further on we shall discuss the reasons which lead us to believe that the looping fibres of rDNA extend into the zone of electron-dense fibrils which surround the fibrillar centre. On a different scale, the loop-like configuration of the nucleolar organizer has been described in somatic plant cells. In these cells, it has been shown that the chromatin of the nucleolar organizer exists in the form of a continuous convoluted loop structure running within the nucleolar body (Godward & Jordan, 1965; La Cour, 1966; La Cour & Wells, 1967; Jordan & Godward, 1969; Chouinard, 1970, 1971). By localizing the nucleolar organizer in the fibrillar centre, our observations confirm the opinion of Goessens (1973, 1974, 1976) based on extensive experimenta- tion in Ehrlich tumour cells. The presence of DNA in the fibrillar centre has been demonstrated by autoradiographic techniques using tritiated actinomycin D and tritiated thymidine (Goessens, 1974, 1976). In the quail oocyte, the presence of a feltwork rich in DNA fibres has been demonstrated in the fibrillar centre using the technique of Cogliati & Gautier (Mirre & Stahl, 1978). At diplotene, the specific relations between the nucleolar chromosomes and fibrillar centres are no longer visible. The nucleolus takes on a reticulated type of structure. Several fibrillar centres, located at variable sites and always surrounded by bands of electron-dense fibrils, can now be seen. This configuration has been described by Chouinard (1971) who interpreted the multiple fibrillar centres as representing cross- or oblique sections of a long contorted loop of chromatin and associated material belonging to a chromosomal nucleolar organizer. The fine structure of the nucleolar fibrillar centre is highly similar to that of the nucleolar organizer described in the secondary constriction of the X chromosome in the Kangaroo rat (Hsu et al. 1967). Goessens & Le Point (1974) have shown that the fibrillar centres of nucleoli in Ehrlich tumour cells persist throughout mitosis in close association with chromosomes.

Fig. 20. Late pachytene oocyte in a 15-day-old mouse embryo. Autoradiography as in preceding figures. The newly formed nucleolus exhibits a fibrillar centre (/c) surrounded by a dense fibrillar zone. Labelling grains are located over the electron-dense fibrils, x 42000. Fig. 21. Pachytene oocyte in a 15-day-old mouse embryo. Autoradiography as in preceding figures. The newly formed nucleolus is at a slightly more advanced stage than in the preceding figure. The fibrillar centre (/c) is unlabelled; the fibrillar component is heavily labelled. Silver grains have not yet appeared over the granular component (g). x 32000. Nucleolar organizer in mouse meiotic oocyte

CE L 31 94 C. Mirre and A. Stahl Observations in somatic cells of plants indicate that the nucleolar organizer presents a fibrillar structure in interphase nuclei (Chouinard, 1970, 1971, 1974, 1975; Lafontaine & Lord, 1969, 1973, 1974)- In Allium porum, the emerging nucleolus has the same ultrastructural aspect as the late anaphase secondary constriction: the incipient nucleolus consists mostly, indeed, of rather densely packed, fine fibrillar material which stains less intensely than the chromosome segments (Lafontaine, 1974; Lafontaine & Lord, 1974). In fibroblasts of the L929 strain, the fibrillar centres contain a fine network of DNA, which stains with oxidized diaminobenzidine and may represent DNA containing the ribosomal genes (Pouchelet, Gansmiiller, Anteunis & Robineaux, 1975; Anteunis, Pouchelet, Gansmiiller & Robineaux, 1975). The presence of DNA in the fibrillar centre, already suggested by Recher, Whitescarver & Briggs (1970), is a fundamental characteristic of this structure since it has been demonstrated in many different cell types in addition to the L929 strain: plant cells (Lafontaine & Lord, 1973; Ryser, Fakan & Braun, 1973; Lord, Nicole & Lafontaine, 1977), Ehrlich tumour cells (Goessens, 1973, 1974, 1976), ovarian follicle cells of Lacerta muralis (Hubert, 1975), quail oocytes (Mirre & Stahl, 1978). In the latter case, in situ hybridization was able to show the presence of rDNA (Knibiehler et al. 1977). Preliminary studies in our laboratory using in situ hybridization indicate that in the mouse oocyte the fibrillar centre also contains rDNA (Stahl et al. 1977). Whether or not the transcription of rDNA occurs in the fibrillar centre is still a controversial point. Recher, Sykes & Chan (1976), although they accept that the fibrils of the fibrillar centres are a dispersed form of chromatin, believe it unlikely that these fibrils are transcribed. Autoradiographic data in our experiments using incorporation of [3H]uridine seem to indicate that the transcription of the ribosomal cistrons essentially takes place at the periphery of the fibrillar centre. Indeed, after brief incorporation, the silver grains are observed at the periphery of the fibrillar centre, especially over the electron-dense fibrils. An hypothetical shift of the preribosomal RNA immediately following its transcription in an internal part of the fibrillar centre is difficult to accept under our experimental conditions since no chase was performed. Moreover, since the studies of Miller & Beatty (1969), it is known that the nascent rRNA does not immediately detach itself from the rDNA, thus allowing observation of transcription units (Trendelenburg, Spring, Scheer & Franke, 1974; Spring et al. 1974; Angelier & Lacroix, 1975; Angelier, 1976). It has generally been accepted that the fibrils of nascent rRNA become detached from the DNA only after 20 minutes. Under such conditions, when the duration of

Fig. 22. Pachytene oocyte in an 18-day-old mouse embryo. Autoradiography following [3H]uridine incorporation for 45 min. The fibrillar and granular components of the nucleolus are labelled; the fibrillar centre (/c) is unlabelled. sy, synaptonemal complex, x 39000. Fig. 23. Diplotene mouse oocyte on postnatal day 3. [3H]uridine incorporation, labelling time 30 min. The fibrillar component of the nucleolus is labelled; no silver grains are seen overlying the fibrillar centres (arrows), x 20500. Nucleolar organizer in mouse meiotic oocyte 95 :§S C. Mirre and A. Stahl

Table i. Variations of [3H]uridine incorporation rate based on silver grain counts in mouse oogonia and oocytes I

Meiosis Stage Mean no. of Early silver grains Oogonia Leptotene Zygotene Pachytene diplotene Nucleolus (n) 6 015 421 Chromosomes (N) 983 ii-77 2779 2389

Ratio njN 061 001 0 00 0 015 O-2I

Fibrillar centre Electron-dense fibrillar zone Fibrillar component Fig. 24. Diagram of the nucleolar-organizer region during nucleolar synthesis. The bivalent is attached to the nuclear envelope. The synaptonemal complex (sc) is surrounded by heterochromatin for about 1-5 /tm. The nucleolar-organizer region is constituted by approximately 50-nm-diameter fibres which emanate from the lateral element of the synaptonemal complex and penetrate into the fibrillar centre. These fibres extend into the dense fibrillar zone which is the site of rDNA transcription. The euchromatic segment located beyond the nucleolar organizer is formed by fibres of about io-o nm diameter. uridine incorporation does not exceed this delay, it should be admitted that the location of the silver grains corresponds to the site of transcription of rDNA. These arguments have led us to propose a diagram for the structure and functioning of the fibrillar centre (Fig. 24). According to this diagram, loops of DNP emanating from the chromosomal secondary constriction penetrate into the fibrillar centre of the nucleolus. These loops extend through the bulk of the fibrillar centre out to its peripheral region. It is in this peripheral zone that the rDNA is transcribed into rRNA. Thus, the constant presence of electron-dense fibrillar strands in immediate contact with the fibrillar centre could be explained. Such fibrillar strands would be composed of both DNA and newly synthesized RNP. The presence of DNA in the layer of dense fibrils seems all the more likely to us since DNA has been demonstrated in the fibrillar zone surrounding the light lacunae in the nucleoli of plant cells (Lafontaine & Lord, 1973). Nucleolar organizer in mouse meiotic oocyte 97 At the onset of nucleolar synthesis during late pachytene, the nucleolus displays natural segregation of its components. Natural segregation in the nucleolus has been described in a variety of cells (for review, see: Smetana & Busch, 1974). This phenomenon was observed in the oocytes of invertebrates, amphibia (Miller, 1966) and of the lizard Lacerta vivipara (Hubert, 1972). In these latter cases, nucleolar segregation coincides with active ribosomal RNA synthesis (Miller & Beatty, 1969; Hubert & Andrivon, 1971). In the mouse spermatocyte, nucleolar formation starts by the appearance of a fibrous component labelled after [3H]uridine incorporation, close to basal knobs at zygotene, followed by the granular component appearing at early pachytene. Nucleolar masses detach from their primary autosomal nucleolar organizer loci during mid-pachytene, migrate toward the sex chromosome masses, rearrange their nucleolar components and form a natural segregated nucleolus which does not incorporate [3H]uridine (Kierszenbaum & Tres, 1974a). In the human spermatocyte at zygotene, the nucleolus is the site of active incorporation of [3H]uridine; at this stage, it is formed by a fibrillar centre separated by dense fibrils from the fibrillo- granular components (Tres, 1975). This nucleolus is band-shaped and strongly resembles that which we described in the mouse oocyte at late pachytene. As pachytene advances in the human spermatocyte, complete nucleolar segregation coincides with a decline of [3H]uridine labelling (Tres, 1975). In the late pachytene quail oocyte, the newly formed nucleolus is composed of a fibrillar centre surrounded by a layer of electron-dense fibrils. Granular components appear later on outside the layer of electron-dense fibrils, in such a way that at a given moment the nucleolus displays three separate zones. Incorporation of [3H]uridine confirms that the fibrils are the precursors of the granular components (Mirre & Stahl, 1976, 1978). In erythrocyte nuclei undergoing reactivation in heterokaryons, the newly formed nucleoli are made of dense fibrillar material or of a fibrillar centre adjacent to a core of densely packed RNA fibrils (Dupuy-Coin, Ege, Bouteille & Ringertz, 1976). It results from these observations that separation of the different components of the nucleolus can be seen during the active phase of nucleolar synthesis as well as during nucleolar inactivation. Distinction between these 2 states of activity can only be achieved by studying the incorporation of an RNA precursor. During synthesis of the nucleolus, continuous formation of preribosomal fibrils at the periphery of the fibrillar centre and progressive transformation of the first-formed fibrils into granules, explains that these components are most often spatially arranged in side-by-side or concentric sheets. Close observation reveals the existence of a fibrillo-granular transition zone between the fibrillar and granular sheets. In cases where segregation is related to inactivation, separation between the fibrillar and granular components is generally more abrupt, with a greater resemblance to that aspect observed following the action of certain drugs such as actinomycin D. In conclusion, all available data point toward the fibrillar centre of the nucleolus as being the region containing the ribosomal cistrons. The latter are carried on the DNP fibres whose origin in the secondary constriction can be demonstrated during pachytene. Study of RNA precursor incorporation suggests that the transcription of rDNA occurs in the peripheral zone of the fibrillar centre, i.e. in the layer of 98 C. Mirre and A. Stahl electron-dense fibrils constantly surrounding the fibrillar centre. If our opinion proves to be correct, the fibrillar centre and the layer of adjacent electron-dense fibrils constitute a single function unit.

This investigation was supported by CNRS (ERA No. 397) and DGRST (No. 75.7.1328).

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