A light- and electron-microscope study of the nucleolus during growth of the oocyte in the prepubertal mouse

In most female mammals which have been investigated, germ cells enter the prophase of meiosis during foetal life and reach the diplotene stage shortly before or immediately after birth (Franchi, Mandi & Zuckerman, 1962; Mauleon, 1967; Peters, 1970). The ensuing diplotene stage (also referred to as the dictyate stage in the case of rodents), during which most of the oocyte growth occurs, is of long duration, since it normally lasts until meiotic division is resumed shortly before ovulation.

In recent years, a number of workers have attempted to define and correlate the integrated and sequential series of ultrastructural changes undergone by the developing mammalian follicular oocyte during the diplotene stage of meiotic prophase (mouse, Yamada, Muta, Motomura & Koga, 1957; Chiquoine, 1960; Parsons, 1962; Rhodin, 1963; Tsuda, 1965; Odor & Blandau, 1969; Wischnitzer, 1970; rat, Sotelo, 1959; Sotelo & Porter, 1959; Odor, 1960; Franchi & Mandi, 1962; hamster, Odor, 1965; Weakley, 1966, 1967, 1969; guinea-pig, Anderson & Beams, 1960; Adams & Hertig, 1964; rabbit, Trujillo-Cenoz & Sotelo, 1959; Blanchette, 1961; Zamboni & Mastroianni, 1966; Anderson, Condon & Sharp, 1970; rhesus monkey, Hope, 1965; man, Wartenberg & Stegner, 1960; Stegner & Wartenberg, 1963; Baca & Zamboni, 1967; Hertig & Adams, 1967).

A survey of these papers reveals, however, that attention has hitherto been confined primarily to isolated phases of mammalian oocyte development, with much emphasis placed on the changes in the ultrastructural organizations of its cytoplasmic organelles and complex surface specializations. Scarcity of knowledge indeed exists concerning the fine-structural changes undergone by the nuclear components of the mammalian oocyte during the same growth period.

It is the purpose of this paper, therefore, to provide a descriptive account of the transformations which occur in the structural organization of the nucleolus during growth of the primary oocyte in the prepubertal mouse.

Litters of ICR albino mice, obtained from Canadian Breeding Farm, St Constant, Province of Quebec, were used. Preliminary observations had revealed that, in prepubertal mice of this strain, growth of a number of oocytes is initiated on the day of birth and completed by postnatal day 14. In order to secure all developmental stages of the growing oocyte in question -in principle, the largest oocytes to be observed in any given ovary at any given time post partum -neonatal mice were sacrificed at daily intervals from birth up to 14 days. All our observations have been made on these growing oocytes. At least 6, but often up to 10 ovaries belonging to animals of the same or different litters were examined at each successive day of life. The animals were killed by decapitation after light ether anaesthesia. The abdomen was rapidly incised and the right ovary of each animal excised and, after being carefully freed from associated adnexa under a dissecting microscope, fixed in toto in an ice-cold 1 % solution of osmium tetroxide in o-i M Sorensen’s phosphate buffer, pH yz, for a 2-h period with occasional agitation. After decanting the fixative, the organ was rapidly dehydrated in a series of increasing concentrations of cold ethanol beginning with 70 % and brought slowly to room temperature in absolute ethanol. Dehydration was completed with 2 additional 15-min changes of absolute ethanol and 2 15-min changes of propylene oxide. Subsequent embedding of the specimens was carried out in Epon 812.

At each postnatal day, the ovaries were studied by means of serial sections for light and electron microscopy. Using a Reichert ultramicrotome and glass knives, the first half portion of each ovary was first ‘thick’ sectioned sagitally to determine, under light microscopy, the size of the largest follicle and contained oocyte present within that ovary. These 0·5–1 μm sections were mounted on slides and stained with borated toluidine blue. Calculation of follicular and oocyte size was performed with a Spencer filar micrometer. Similar thick serial sections were also cut from the second half portion of each ovary until the nucleus of one amongst the largest follicular oocytes was reached. At this time, the block was carefully trimmed and the selected oocyte, together with the surrounding follicular cells, were serially sectioned for electron microscopy. Serial sectioning for light microscopy was then resumed and continued until the nucleus of another selected follicular oocyte appeared. Alternate serial sectioning for light and electron microscopy was continued throughout most of the remaining ovarian mass. It should be stressed at this point that, at each postnatal day, the oocytes and follicles selected for study were among the largest present in the ovary and that none showed any visible sign of involution or atresia. Thus, it can be confidently assumed that these oocytes were, at the time of fixation, in a normal and active state of growth and differentiation.

For electron microscopy, sections displaying silver-to-pale-gold interference colours were picked up on uncoated 200-mesh copper grids. The sections were stained with uranyl acetate for 5 min, followed by lead citrate for 10 min, and examined in a Siemens Elmiskop IA electron microscope using the double condenser, 80 kV and 5o μ m objective aperture.

Preparations for light microscopy were studied with a Leitz binocular microscope, using a ribbon-filament lamp, Köhler illumination, and a 100 ×; 1·32 N.A. apochromatic objective. An orange Wratten filter was used in the illumination system.

For descriptive purposes, the growth period of the oocytes studied -i.e. those in which growth is initiated on the day of birth and completed by postnatal day 14 -will be divided into 3 successive stages depending on the extent of follicle development, and these will be referred to as the unilaminar, the bilaminar and the plurilaminar follicle stages.

During this stage, the growing oocyte is surrounded by a single complete layer of follicle cells. On postnatal day 1, the largest oocytes, already in the dictyate stage of meiotic prophase, are enclosed by a layer of flattened follicle cells. The oocyte is then 20 μm in diameter, while its rounded nucleus measures up to 12 μm. On postnatal days 2 and 3, the follicle cells increase in number and a change in their shape from flattened to rectangular to cuboidal occurs. By postnatal day 4, most of the follicle cells encircling the largest oocytes exhibit a low columnar configuration; the oocyte has then grown into a 40-μm sphere containing a more-or-less centrally located nucleus about 16 μm in diameter.

Gradual transition from a unilaminar follicle with low columnar cells to one containing 2 layers of isodiametric follicle cells occurs during postnatal days 5 and 6. Such a transition is at first characterized by the appearance of small semi-prismatic cells in between the low columnar cells. A further increase through mitosis in the number of such small cells over those present at this transitional phase seemingly results in a bilaminar follicle. On postnatal days 7 and 8, the largest oocytes are seen to be wrapped with 2 distinct layers of tightly packed follicle cells. By postnatal day 8, the largest oocytes are spheroidal, measure up to 50 μm in diameter and contain a centrally or paracentrally located, rounded nucleus approximately 18 μm in diameter.

By the ninth or tenth postnatal day, 3 and in places 4 layers of rounded, rapidly proliferating follicle cells are seen encircling the oocyte. Already at that time, tiny irregularly shaped spaces, presumably filled with follicular fluid, are recognizable amongst the follicle cells. On postnatal days 11 and 12, the largest oocytes are seen to be wrapped with 4 to 6 layers of still actively dividing follicle cells which tend to assume a rounded shape. The small follicular fluid-containing spaces have by now fused into larger cleft-shaped or rounded pools located roughly halfway between the periphery of the oocyte and the border of the follicle. In the course of postnatal days 13 and 14, the pools of follicular fluid open into each other thus giving rise to a single small crescentic cavity, the antrum; the largest oocytes are then encircled by 8 to 10 layers of rounded follicle cells. By the time the largest follicles (230–250 μm in diameter) acquire their small antrum, the contained oocyte has attained its full size, i.e. approximately 70μm in diameter; the more-or-less centrally located nucleus has then reached a diameter of about 22 μm. The nucleus apparently ceases growing when the oocyte reaches its maximum size. To summarize, during the period extending from birth through postnatal day 14, the diameter of the growing mouse oocyte increases from 20 to 70 μm while that of its nucleus increases from 12 to 22 μm; the 43-fold increase in oocyte volume is therefore accompanied by a 6-fold increase in nuclear volume.

The nucleus of the growing mouse oocyte contains a single large nucleolus and, quite often, one or two smaller ones which lie widely separated from one another; all of these nucleoli assume a rounded shape. As already noted by several authors (Yamada et al. 1957; Parsons, 1962; Tsuda, 1965; Odor & Blandau, 1969), the diplotene (or dictyate) bivalents of the growing mouse oocyte, very likely as a result of their highly unravelled condition, are not recognizable as such within the nucleus either under light or electron microscopy. Besides the nucleolus, the mouse oocyte nucleus exhibits a number of smaller, rounded bodies stained with toluidine blue and Feulgen-negative, which become especially conspicuous during the bilaminar and the plurilaminar follicle stages; 3 ultrastructurally distinct types of such extranucleolar bodies have been identified so far (Chouinard, 1970a).

On the first postnatal day, the densely stained nucleolus, 2–2·5μm in diameter, appears quite homogeneous in internal structure under light microscopy (Fig. 1). Favourable sections reveal the presence of a sizeable mass of chromatin material, moderately stained with toluidine blue and also Feulgen-positive, anchored to the nuclear envelope and intimately associated, on one side, with the nucleolar surface (Fig. 1). Such a condensed mass of nucleolus-associated chromatin (nac) is possibly best considered as representing the heterochromatic centromeric end (‘basal knob’) of one or perhaps several of the bivalents known to be associated with nucleolar formation in the species investigated (Ohno, Kaplan & Kinosita, 1957; Woollam & Ford, 1964; Woollam, Millen & Ford, 1967; Sugihara & Yasuzumi, 1970). On postnatal days 2 and 3, the nucleolus is seen to contain a number of small, scattered clumps of a material which stains slightly more intensely than the rest of the nucleolar mass (Figs. 2, 3). During the same period, the nucleolus-associated chromatin disappears gradually from view, but the nucleolus continues to occupy an eccentric position within the nuclear cavity (Fig. 3). By postnatal day 4, the nucleolus has grown into a spheroidal body, 5–6 μm in diameter, which exhibits a still more heterogeneous appearance. In addition to the small scattered clumps of more intensely stained material described previously, the nucleolus is indeed seen to contain numerous barely resolvable unstained spaces distributed more-or-less evenly within its mass (Fig. 4).

Throughout the unilaminar follicle stage (Figs. 17–20), the oocyte nucleolus shows, under electron microscopy, an overall reticulated-type of structure consisting of 4 ultrastructurally and topographically definable components which, for descriptive purposes, will be referred to as components A, B, C and D. Component A(a) exhibits a moderately electron-dense fibrillogranular texture and is seen to be made up of rather tightly arranged convoluted fibrillar elements, ranging from 6 to 10 nm in width, randomly intermingled with more electron-dense granules approximately 15 nm in diameter. In all nucleolar profiles examined, this fibrillogranular component occupies most parts of the reticulated framework. Because of its abundance and rather uniform distribution, the component in question has the appearance of a matrix in which the other structural components (B, C and D) of the nucleolus are embedded. Component B(b) is of low electron opacity and consists of loosely dispersed and ill-defined fibrillar elements indistinguishable from those present in the surrounding nucleoplasm. This component fills the numerous electron-transparent interstices of the reticulated nucleolar framework. In some nucleolar profiles, the interstices are rounded or slightly elongated, but in others they are much more irregular in shape and appear to form some sort of canalicular network. It is of interest to note also that in a number of nucleolar profiles, structural continuity between the content of some of the more peripherally located interstices and the surrounding nucleoplasm is observed (Figs. 17, 18, arrows). Component C(c), the most electron-opaque structural element of the nucleolar mass, is made up of closely packed convoluted fibrils, 6-10 nm in diameter, possibly with an intervening, seemingly amorphous, matrical substance. All nucleolar profiles examined show this component to be present in the peripheral portion of a number of small rounded or slightly elongated fibrillar islands quite widely and evenly distributed within the nucleolar mass. Such fibrillar islands, with a diameter of 0·5–0·8 μm, are bordered, in part, by elements of the fibrillogranular framework with which they blend more or less imperceptibly, and, in part, by some of the electron-transparent interstices. Component D (d) is only slightly less electron-dense than component C and consists exclusively of closely arranged fibrillar elements 6-10 nm in width. This component is invariably seen to occupy the more-or-less central portion of the previously described fibrillar islands scattered within the nucleolar mass. A characteristic feature of component D is, therefore, to be more-or-less completely enclosed or walled off by the more electron-dense fibrillar material of component C. In all nucleolar profiles examined, the small areas occupied by component D present an outline that tends to be rounded or slightly elongated. Because of their central location and fibrillar texture, these nucleolar areas containing component D will be referred to as ‘fibrillar centres’, a term recently coined by Recher, Whitescarver & Briggs (1969) to describe comparable areas in nucleoli of human tissue culture cells. Judging from their size and topographical distribution, there can be little doubt that the larger fibrillar islands just described correspond to the more intensely stained clumps of material observed within the nucleolar mass in the light microscope (Figs. 2–4). The nucleolus-associated chromatin (nac) appears slightly more electron-dense than the nucleoplasm and is seen to consist of rather loosely knit fibrillar elements 6–10 nm in width.

Visual comparison of nucleolar profiles reveals that rapid growth of the nucleolar mass during the unilaminar follicle stage is brought about mainly through an increase in the overall dimensions of the fibrillogranular framework accompanied by a parallel increase in the number and also, but to a smaller extent, the size of its electron-transparent interstices. On postnatal day 4, some of these interstices indeed become resolvable in the light microscope (Fig. 4). An increase in the number and possibly also the size of some of the fibrillar islands with their contained fibrillar centres also contributes, but to a much smaller extent, to the overall growth of the nucleolar mass during the same period.

During the transition from the uni-to the bilaminar follicle stage (postnatal days 5 and 6), most of the previously described barely resolvable unstained spaces observed within the nucleolar mass, by light microscopy, gradually disappear and a number of small but clearly recognizable rounded unstained areas, designated from now on as nucleolar vacuoles, make their appearance within the otherwise densely and uniformly stained nucleolar body (Figs. 5, 6). During postnatal days 7 and 8, there occurs a gradual increase in size of several of these vacuoles, some eventually reaching up to 1·5–m in diameter (Figs. 7, 8). Favourably transected nucleoli also reveal the existence of a barely definable core of stainable material within these enlarging vacuoles (Figs. 7, 8, arrows). By the end of the bilaminar follicle stage (postnatal day 8), the nucleolus has grown into a vacuolated but otherwise densely and homogeneously stained spherical body which may reach up to 7 –m in diameter (Fig. 8). There can be little doubt that the appearance and subsequent enlargement of the nucleolar vacuoles contribute significantly to the increase in volume of the nucleolar mass during the bilaminar follicle stage.

On postnatal day 5, the nucleolus continues to exhibit, under electron microscopy, an overall reticulated-type of structure consisting of (1) a moderately electron-dense fibrillogranular framework, (2) numerous and evenly distributed electron-transparent interstices, of varying size and shape, filled with a material whose texture resembles that of the nucleoplasm, and (3) a number of small widely scattered pleomorphic islands usually made up of an outer slightly more electron-dense and an inner less electron-dense (‘fibrillar centre’) fibrillar material (Fig. 21). In the course of postnatal days 6 and 7, several profound ultrastructural and organizational changes take place more or less concomitantly within the growing nucleolar mass (Figs. 22-24). The main features of such intranucleolar transformations are outlined as follows: (1) Most parts of the moderately electron-dense fibrillogranular framework gradually become predominantly fibrillar in texture as a result of what appears to be an unravelling or unfolding of its constituent 15-nm granules. A portion of a nucleolus undergoing such a degranulation process is depicted in Fig. 22. (2) As the nucleolus undergoes gradual degranulation, the outer slightly more electron-dense regions of the previously described fibrillar islands become no longer recognizable as such; the inner region (‘fibrillar centres’) of the same islands, however, apparently persist as rounded fibrillar areas but only slightly less electron-dense than the rest of the stainable portion of the nucleolar mass (Fig. 22). (3) The light-staining interstices decrease rapidly both in size and number as a result of what appears to be the gradual accumulation within their interior of a fibrillar material matching in density that present in the nucleolar framework itself; remnants of these interstices are represented by small, rounded, lightly stained spaces containing nucleoplasm-like material (Figs. 23, 24). (4) As the nucleolar mass assumes a more compact appearance, other rounded, lightly stained spaces of varying size and shape and corresponding to the forming nucleolar vacuoles, appear in the regions formerly occupied by some of the fibrillar islands and their bordering interstices (Figs. 23, 24). It is of interest that small rounded patches of nucleolar material usually located adjacent to these nucleolar vacuoles retain for a while their original fibrillogranular texture (Fig. 24). Examination of a number of favourably transected nucleoli also reveals the presence within the emerging nucleolar vacuoles of a centrally or paracentrally located core of fibrillar material of variable size and electron density and which is thought to correspond, at least in part, to the nucleolar areas previously designated as fibrillar centres (Figs. 23, 24). The remaining portion of the nucleolar vacuoles is occupied by a fibrillar material, the texture and density of which resemble that of the nucleoplasm.

By postnatal day 8, the dense portion of the nucleolar mass is seen to consist, for the most part, of an intricate feltwork of convoluted fibrillar elements, 6-10 nm in diameter, lying in what appears to be a dense ill-defined amorphous matrix (Figs. 25, 26). Small rounded patches of dense fibrillogranular material are still occasionally seen located adjacent to the vacuolar space (Fig. 25). The dense portion of the nucleolar mass continues to be permeated by a number of tiny electron-transparent spaces, undoubtedly remnants of the previously described nucleolar interstices (Figs. 25, 26). Favourable sections invariably show the presence of a fibrillar core, of variable size and electron opacity, within the enlarging nucleolar vacuoles (Figs. 25, 26). The remaining portion of the vacuolar space is occupied by a material, the texture and electron density of which match quite closely that of the nucleoplasm and by small, scattered and ill-defined clumps of material tinctorially quite similar to that observed within the vacuolar core itself (Figs. 25, 26). Occasionally, narrow communication channels between adjacent nucleolar vacuoles and between nucleolar vacuoles and the surrounding nucleoplasm are observed.

During transition from the bi-to the plurilaminar follicle stage (postnatal days 9 and 10), all nucleolar profiles display, in the light microscope, several rounded vacuolar areas, of varying size, and partially filled with a material which stains only slightly less intensely than the rest of the nucleolar mass (Figs. 9, 10). In the course of postnatal days 11 and 12, these nucleolar vacuoles, very likely as a result of fusion, become fewer in number but larger in size, some eventually reaching up to 3 μm in diameter. As the vacuoles grow larger, they also become almost completely filled with stainable material (Figs. 11, 12). During postnatal days 12 and 13, the contours of the nucleolar vacuoles in question become increasingly difficult to delineate because the stainability of their content comes gradually to match that of the rest of the nucleolar mass (Figs. 13, 14). By the end of the growth period of the oocyte (postnatal day 14), the nucleolus has grown into a densely and uniformly stained spherical body, which may reach up to 9μm in diameter (Figs. 15,16).

Between postnatal days 9 and 12, the nucleolus appears, under electron microscopy, in the form of a continuous electron-dense mass in which are embedded numerous tiny and evenly distributed electron-transparent areas (remnants of the nucleolar interstices), as well as a number of much larger rounded spaces (nucleolar vacuoles) containing a nucleoplasm-like substance and variable amounts of a moderately electron-dense fibrillogranular material (Figs. 27–30). The electron-dense portion of the nucleolar mass is made up of a feltwork of closely packed and randomly oriented fibrils, 6-10 nm in diameter, apparently immersed in an amorphous matrical substance not readily analysed in the micrographs. The nucleolar interstices contain a loosely dispersed fibrillar material matching that of the nucleoplasm in density. The moderately electron-dense material that gradually accumulates within the vacuole interior consists of intermingled masses made up of closely arranged fibrillar elements, 6–10 nm in width, interspersed with electron-dense granules of ribosomal dimensions (Figs. 27–30). Favourable sections reveal that, in places at the periphery of the vacuolar spaces, the contained fibrillogranular material blends more-or-less imperceptibly with the surrounding dense fibrillar material of the nucleolar mass (Figs. 27–30, arrows). The previously described intravacuolar fibrillar core becomes no longer recognizable as such as soon as the fibrillogranular material begins to accumulate within the interior. In the course of postnatal days 12 and 13, the lightly stained nucleolar interstices disappear gradually from view and the accumulated fibrillogranular material of the nucleolar vacuoles becomes transformed into a compact substance, exclusively fibrillar in texture, and eventually matching the rest of the nucleolar mass in electron density (Fig. 31). The degranulation process of the fibrillogranular material contained within the nucleolar vacuoles bears strong resemblance to that which also takes place in the fibrillogranular framework of the same nucleolus during the bilaminar follicle stage (compare Fig. 22).

By postnatal day 14, the prominent nucleolus appears as a dense compact mass exhibiting no internal structural organization (Fig. 32). The fine texture of the nucleolar material, because of its compactness, is not readily analysed in ordinary pale-gold sections examined under electron microscopy. However, grey-silver sections provide enough transparency to resolve the texture of this apparently homogeneous material into a bewildering array of minute punctate and linear profiles of varying size and density (Fig. 32, inset). This characteristic appearance is probably best interpreted as resulting from the longitudinal and transverse sectioning of a feltwork of randomly oriented fibrils which are 6–10 nm in diameter. The punctate profiles would then represent fibrils seen in transverse sections, since they have the same size range as the thicknesses of linear profiles. In order to account for the relatively high electron opacity of the mature oocyte nucleolus, it appears more than likely that the feltwork of nucleolar fibrils is also permeated by some sort of amorphous matrical substance not readily characterized in the micrographs.

Our observations reveal that the nucleolus undergoes a gradual increase in size and a precise pattern of changes in internal structure that can be correlated with oocyte growth and follicle development in the prepubertal mouse. In the following discussion, an attempt will be made to interpret these morphological changes, at each stage of follicular oocyte development, in the light of current knowledge concerning the architectural and functional organization of the nucleolus in general.

The occurrence of a fibrillogranular framework, interstices and fibrillar islands within the nucleolar mass has been reported in a number of previous electron microscope studies of the nucleolus and in a wide variety of eukaryotic cells, although the designation of these various nucleolar components has differed greatly (see Vincent & Miller, 1966; Bimstiel, 1967; Hay, 1968; Bernhard & Granboulan, 1968; Busch & Smetana, 1970). The further subdivision of the fibrillar islands of the nucleolar mass into an outer more electron-dense and an inner less electron-dense area has also been recorded by several authors in recent years and in a variety of cell types (Schoell, 1964; Yasuzumi & Sugihara, 1965; Terzakis, 1965; Jezequel, Seeve & Steiner, 1967; Recher et al. 1969; Recher, Whitescarver & Briggs, 1970; Shinozuka, 1970; Simard, 1970; Hardin, Spicer & Malanos, 1970). The inner, less electron-dense areas (‘fibrillar centres’ of Recher et al. 1969) of the fibrillar islands have usually been considered as a distinct structural component of the nucleolus, rich in pepsin-digestible protein and RNA; also, the possibility of DNA being present in such fibrillar centres has not been excluded (Recher et al. 1969, 1970).

On the basis of some of our observations, and in the light of accumulated knowledge concerning the spatial relationship of these various nucleolar components to the nucleolar organizing region (NOR) of the nucleolar chromosome in general (see reviews by Kopac & Mateyko, 1964; Bimstiel, 1967; Busch & Smetana, 1970), it is tempting to postulate that the so-called fibrillar centres seen in all nucleolar profiles of the growing mouse oocyte, instead of being discrete entities, correspond, in fact, to cross- or oblique sections of a long contorted loop of chromatin and associated material belonging to the nucleolar organizing segment of the nucleolar chromosome(s). In our material, support for such a suggestion would come mainly from the following observations and considerations, (a) Throughout the unilaminar follicle stage, the content of the fibrillar centres is seen to exhibit an electron density matching quite closely that of the more condensed regions of the chromatin mass associated with the nucleolar surface on postnatal days 1 and 2 (Figs. 17,18). (6) Removal of intranucleolar DNA enzymically from formaldehyde-fixed material (Chouinard, 1970b) induces a noticeable reduction in heavy metal uptake by both the fibrillar centres and the nucleolus-associated chromatin of the oocyte, (c) On postnatal days 1 and 2, favourable sections of the nucleolus reveal structural continuity between the content of some of the fibrillar centres and the fibrillar content of the nucleolus-associated chromatin. (d) As the nucleolus enlarges and the number of fibrillar centres per nucleolar profile increases, the mass of nucleolus-associated chromatin decreases in size and eventually disappears, thus suggesting a gradual incorporation of at least part of this chromatin (allegedly resting nucleolar organizing chromatin of the nucleolar chromosome(s)) into the fibrillar centres of the growing nucleolar body, (e) The fibrillar centres, supposedly containing chromatin material, are invariably seen to be intimately associated with the dense fibrillar component of the nucleolar mass, as seems to be the case for intranucleolar chromatin in most other investigated types of nucleoli in eukaryotic cells, including oocytes (Miller, 1966; Birnstiel, 1967; Hay, 1968; Miller & Beatty, 1969; Perry, 1969; Busch & Smetana, 1970).

On the basis of our observations, a tridimensional reconstruction of the growing oocyte nucleolus, during the unilaminar follicle stage, would very likely reveal that the intranucleolar interstices are, in fact, part of an intricate system of intercommunicating channels filled, at least in part, with nucleoplasm. Such a system undoubtedly plays an essential role in nucleolar growth by increasing the surfaces of functional exchanges and by facilitating the transport and accumulation of materials from both intra- and extranucleolar origin.

Since our observations provide no evidence for the release of the granular elements of the nucleolus into the surrounding nucleoplasm, the degranulation process of the nucleolar mass is best interpreted in terms of conversion of the 15-nm granules into fibrils through unravelling or unfolding. The observations of Marinozzi (1964) and Smetana, Unuma & Busch (1968), on the 2 basic structural components (fibrils and granules) of the nucleolus, clearly indicate that the 15-nm granules are, indeed, wrapped-up fibrils and that transitional configuration forms frequently occur. There is also strong evidence that the nucleolar fibrils are the precursors of the nucleolar 15-nm particles (Granboulan & Granboulan, 1965; Geuskens & Bernhard, 1966). Conversely, degranulation of the nucleolar mass through unravelling of its granular components has also been produced experimentally following exposure of cultured animal cells to supranormal temperature; such a process has also been shown to be reversible (Simard & Bernhard, 1967; Simard, 1970).

A 3-dimensional reconstruction of the oocyte nucleolus would very likely reveal here also that the nucleolar vacuoles are not isolated spaces, but part of an intranucleolar system of intercommunicating cavities of varying size filled, at least in part, with material of the nucleoplasm. It is conceivable that such a vacuolar system serves primarily as a means of increasing the surfaces of functional exchanges and facilitating the transport of materials from both intra- and extranucleolar sources.

Although the origin of the vacuolar cores remains somewhat conjectural on the basis of our present micrographs, one is tempted to suggest that they are derived from the previously described fibrillar centres, since the nucleolar vacuoles arise at some of the sites formerly occupied by the fibrillar areas and contained fibrillar centres of the nucleolar mass. The vacuolar cores, like the previously discussed fibrillar centres, could then be tentatively considered as corresponding to cross- or oblique sections of the nucleolar organizing segment(s) of the nucleolar chromosome(s). It is conceivable also that the vacuolar cores are but the morphological manifestation of a phenomenon related to rDNA amplification. Such a phenomenon has recently been shown to occur not only in multinucleolate, but also in uninucleolate oocytes (Brown & Dawid, 1969; Dawid & Brown, 1970).

The present study being essentially morphological, no definite conclusion can be drawn concerning the origin of the fibrillogranular material that accumulates inside the nucleolar vacuoles. In relation to this problem, 2 possibilities, which are not mutually exclusive, should, nevertheless, be envisaged. One would be that most, if not all, of this material simply represents the accumulated products of the functional activity of the rest of the nucleolar mass. The frequent observation that the material located in the peripheral portion of the vacuoles merges more-or-less imperceptibly with that of the nucleolar body proper would be consistent with such a view. Most of the previous studies on nucleolar vacuoles in both plant and animal cells also suggest that these structures indeed represent sites of accumulation rather than sites of synthesis of their contained material (Esper, 1965; Barlow, 1970). Another possibility would be that the nucleolar vacuoles themselves are the sites of synthesis of at least part of their contained material. It is conceivable, for instance, that the core of fibrillar material shown to be present within such vacuoles is somehow instrumental in the synthesis of vacuolar material. Such an interpretation would be considerably strengthened if future observations confirm that the vacuolar cores contain the intra-nucleolar chromatin. Of interest in this regard are the observations of Love & Walsh (1968), suggesting that the vacuolar (‘nucleolinar’) RNA is intimately associated with DNA -even if the vacuoles do not contain any stainable DNA.

The fibrillar appearance of the nucleolus toward the end of mammahan oogenesis has already been noted by Parsons (1962) and Adams & Hertig (1964). In the fully grown mammalian oocyte, the fibrillar content of the nucleolus constitutes very likely a stable storage form for the accumulated nucleolar material (Hay, 1968).

In summary, ah of our observations would be consistent with the generally held view that the nucleolus, during growth of the primary oocyte, is the site of massive synthesis and storage of nucleolar material (Vincent, Baltus, Lovlie & Mundell, 1966; Oakberg, 1967, 1968; Hay, 1968; Baker, Beaumont & Franchi, 1969; Brown & Dawid, 1969). The material stored within the dormant oocyte nucleoli is released to the cytoplasm only later on, at the time of nuclear envelope breakdown, and eventually utilized during early embryogenesis (Davidson, Crippa, Allfrey & Mirsky, 1966; Crippa, Davidson & Mirsky, 1967; Davidson, 1968).

This work was supported by a grant (MA-770) from the Medical Research Council of Canada.