A LIGHT- AND ELECTRON-MICROSCOPE STUDY OF THE NUCLEOLUS DURING GROWTH … · 2005. 8. 20. · J....

J. Cell Sci. 9 ) 637-663 (i97i) 637

Printed in Great Britain

A LIGHT- AND ELECTRON-MICROSCOPE

STUDY OF THE NUCLEOLUS DURING

GROWTH OF THE OOCYTE IN THE

PREPUBERTAL MOUSE

L. A. CHOUINARD

Department of Anatomy, Laval University, Quebec City, Canada

SUMMARY

The ordered changes which occur in the structural organization of the nucleolus duringgrowth of the mouse oocyte have been studied by both light and electron microscopy. Allobservations have been made on those oocytes whose growth is initiated on the day of birthand completed by postnatal day 14 in prepubertal animals of the JCR albino mouse strain.During that period the oocyte nucleolus undergoes an approximate 90-fold increase in volume.During the unilaminar follicle stage (from birth to postnatal day 4), the growing nucleolusexhibits an overall reticulated-type of structure consisting of: (1) a moderately electron-densefibrillogranular component occupying most parts of the nucleolar framework; (2) an electron-transparent nucleoplasm-like component filling the numerous interstices of the nucleolarframework; (3) an electron-dense fibrillar component located in the peripheral portion of anumber of small islands widely and uniformly scattered within the nucleolar framework, and(4) a slightly less-dense fibrillar component situated in the central portion of these same islandsand referred to as fibrillar centres. Increase in nucleolar volume during that stage is broughtabout mainly through an increase in the overall dimensions of the fibrillogranular framework,accompanied by a parallel increase in the number and, to a certain extent, the size of its electron-transparent interstices. During the bilaminar follicle stage (postnatal day 5 through 8), thefollowing structural and organizational changes take place more or less concomitantly withinthe still enlarging nucleolar mass: (1) the fibrillogranular framework becomes predominantlyfibrillar in texture as a result of what appears to be an unravelling or unfolding of its consti-tuent granules of ribosomal dimensions; (2) the nucleolar interstices decrease rapidly both innumber and size because of the accumulation within their interior of a material the texture anddensity of which match that present in the nucleolar framework itself; and (3) a number ofrounded electron-transparent spaces, the nucleolar vacuoles, make their appearance in theregions formerly occupied by some of the fibrillar islands and adjacent interstices. Increase innucleolar volume during that stage is largely due to the appearance and subsequent enlarge-ment of the nucleolar vacuoles in question. During the plurilaminar follicle stage (postnatalday 9 through 14), the following sequential events take place within the nucleolar mass: (1) amoderately electron-dense fibrillogranular material accumulates within the nucleolar vacuoles;(2) this fibrillogranular material, which eventually fills all vacuolar spaces, undergoes de-granulation and a concomitant increase in density, eventually matching that of the rest of thenucleolar mass; (3) all remnants of the lightly stained nucleolar interstices disappear from view;and (4) the fully grown rounded nucleolus finally appears as a dense, compact mass, exclusivelyfibrillar in texture, and exhibiting no internal structural organization.

An attempt is made to interpret these changes in the light of current knowledge concerningthe architectural and functional organization of the mammalian nucleolus in general. Theobservations are consistent with the view that the nucleolus, during growth of the primaryoocyte, is the site of massive synthesis and storage of nucleolar material.

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638 L. A. Chouinard

INTRODUCTION

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

In recent years, a number of workers have attempted to define and correlate theintegrated and sequential series of ultrastructural changes undergone by the develop-ing mammalian foUicular oocyte during the diplotene stage of meiotic prophase(mouse, Yamada, Muta, Motomura & Koga, 1957; Chiquoine, i960; Parsons, 1962;Rhodin, 1963; Tsuda, 1965; Odor & Blandau, 1969; Wischnitzer, 1970; rat, Sotelo,1959; Sotelo & Porter, 1959; Odor, i960; Franchi & Mandl, 1962; hamster, Odor,1965; Weakley, 1966, 1967, 1969; guinea-pig, Anderson & Beams, i960; Adams &Hertig, 1964; rabbit, Trujillo-Cenoz & Sotelo, 1959; Blanchette, 1961; Zamboni &Mastroianni, 1966; Anderson, Condon & Sharp, 1970; rhesus monkey, Hope, 1965;man, Wartenberg & Stegner, i960; Stegner & Wartenberg, 1963; Baca & Zamboni,1967; Hertig & Adams, 1967).

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

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

MATERIALS AND METHODSLitters 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 thisstrain, growth of a number of oocytes is initiated on the day of birth and completed by postnatalday 14. In order to secure all developmental stages of the growing oocyte in question - inprinciple, 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 observationshave been made on these growing oocytes. At least 6, but often up to 10 ovaries belonging toanimals of the same or different litters were examined at each successive day of life. Theanimals were killed by decapitation after light ether anaesthesia. The abdomen was rapidlyincised and the right ovary of each animal excised and, after being carefully freed fromassociated adnexa under a dissecting microscope, fixed in toto in an ice-cold 1 % solution ofosmium tetroxide in o-i M Sorensen's phosphate buffer, pH 7'2, for a 2-h period with occasionalagitation. After decanting the fixative, the organ was rapidly dehydrated in a series of increasingconcentrations of cold ethanol beginning with 70 % and brought slowly to room temperaturein absolute ethanol. Dehydration was completed with 2 additional 15-min changes of absoluteethanol and 2 15-min changes of propylene oxide. Subsequent embedding of the specimenswas carried out in Epon 812.

Nucleolus in growing mouse oocyte 639

At each postnatal day, the ovaries were studied by means of serial sections for light andelectron microscopy. Using a Reichert ultramicrotome and glass knives, the first half portion ofeach ovary was first 'thick' sectioned sagitally to determine, under light microscopy, the sizeof the largest follicle and contained oocyte present within that ovary. These 0-5-1 fim sectionswere mounted on slides and stained with borated toluidine blue. Calculation of follicular andoocyte size was performed with a Spencer filar micrometer. Similar thick serial sections werealso cut from the second half portion of each ovary until the nucleus of one amongst the largestfollicular oocytes was reached. At this time, the block was carefully trimmed and the selectedoocyte, together with the surrounding follicular cells, were serially sectioned for electronmicroscopy. Serial sectioning for light microscopy was then resumed and continued until thenucleus of another selected follicular oocyte appeared. Alternate serial sectioning for light andelectron microscopy was continued throughout most of the remaining ovarian mass. It shouldbe stressed at this point that, at each postnatal day, the oocytes and follicles selected for studywere among the largest present in the ovary and that none showed any visible sign of involutionor 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 werepicked up on uncoated 200-mesh copper grids. The sections were stained with uranyl acetatefor 5 min, followed by lead citrate for 10 min, and examined in a Siemens Elmiskop IA electronmicroscope using the double condenser, 80 kV and 50-fim objective aperture.

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

OBSERVATIONS

Oocyte growth and follicle development

For descriptive purposes, the growth period of the oocytes studied - i.e. those inwhich growth is initiated on the day of birth and completed by postnatal day 14 - willbe 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 plurilaminarfollicle stages.

The unilaminar follicle stage. During this stage, the growing oocyte is surrounded bya single complete layer of follicle cells. On postnatal day 1, the largest oocytes, alreadyin the dictyate stage of meiotic prophase, are enclosed by a layer of flattened folliclecells. The oocyte is then 20 /im in diameter, while its rounded nucleus measures upto 12 /im. On postnatal days 2 and 3, the follicle cells increase in number and a changein 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 con-figuration ; the oocyte has then grown into a 40-/011 sphere containing a more-or-lesscentrally located nucleus about 16/im in diameter.

The bilaminar follicle stage. Gradual transition from a unilaminar follicle with lowcolumnar cells to one containing 2 layers of isodiametric follicle cells occurs duringpostnatal days 5 and 6. Such a transition is at first characterized by the appearance ofsmall semi-prismatic cells in between the low columnar cells. A further increasethrough mitosis in the number of such small cells over those present at this transitionalphase seemingly results in a bilaminar follicle. On postnatal days 7 and 8, the largestoocytes 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 /tm in diameter

640 L. A. Chouinard

and contain a centrally or paracentrally located, rounded nucleus approximately 18 fimin diameter.

The plurilaminar follicle stage. By the ninth or tenth postnatal day, 3 and in places4 layers of rounded, rapidly proliferating follicle cells are seen encircling the oocyte.Already at that time, tiny irregularly shaped spaces, presumably filled with follicularfluid, are recognizable amongst the follicle cells. On postnatal days 11 and 12, thelargest oocytes are seen to be wrapped with 4 to 6 layers of still actively dividingfollicle cells which tend to assume a rounded shape. The small follicular fluid-con-taining spaces have by now fused into larger cleft-shaped or rounded pools locatedroughly 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 eachother thus giving rise to a single small crescentic cavity, the antrum; the largestoocytes are then encircled by 8 to 10 layers of rounded follicle cells. By the time thelargest follicles (230-250 /tm in diameter) acquire their small antrum, the containedoocyte has attained its full size, i.e. approximately 70/nn in diameter; the more-or-less centrally located nucleus has then reached a diameter of about 22 fim. The nucleusapparently ceases growing when the oocyte reaches its maximum size. To summarize,during the period extending from birth through postnatal day 14, the diameter of thegrowing mouse oocyte increases from 20 to 70 /tm while that of its nucleus increasesfrom 12 to 22 fim; the 43-fold increase in oocyte volume is therefore accompanied bya 6-fold increase in nuclear volume.

The nucleolus during growth of the oocyte

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; allof these nucleoli assume a rounded shape. As already noted by several authors(Yamadaef al. 1957; Parsons, 1962; Tsuda, 1965; Odor & Blandau, 1969), the diplo-tene (or dictyate) bivalents of the growing mouse oocyte, very likely as a result of theirhighly unravelled condition, are not recognizable as such within the nucleus eitherunder light or electron microscopy. Besides the nucleolus, the mouse oocyte nucleusexhibits a number of smaller, rounded bodies stained with toluidine blue and Feulgen-negative, which become especially conspicuous during the bilaminar and the pluri-laminar follicle stages; 3 ultrastructurally distinct types of such extranucleolar bodieshave been identified so far (Chouinard, 1970a).

The unilaminar follicle stage. On the first postnatal day, the densely stained nucle-olus, 2-2-5 lxrn m diameter, appears quite homogeneous in internal structure underlight microscopy (Fig. 1). Favourable sections reveal the presence of a sizeable massof chromatin material, moderately stained with toluidine blue and also Feulgen-positive, anchored to the nuclear envelope and intimately associated, on one side, withthe nucleolar surface (Fig. 1). Such a condensed mass of nucleolus-associated chro-matin (nac) is possibly best considered as representing the heterochromatic centro-meric end ('basal knob') of one or perhaps several of the bivalents known to beassociated 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 numberof small, scattered clumps of a material which stains slightly more intensely than therest of the nucleolar mass (Figs. 2, 3). During the same period, the nucleolus-associ-ated chromatin disappears gradually from view, but the nucleolus continues tooccupy an eccentric position within the nuclear cavity (Fig. 3). By postnatal day 4,the nucleolus has grown into a spheroidal body, 5-6 /tm in diameter, which exhibitsa still more heterogeneous appearance. In addition to the small scattered clumps ofmore intensely stained material described previously, the nucleolus is indeed seen tocontain numerous barely resolvable unstained spaces distributed more-or-less evenlywithin 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 of4 ultrastructurally and topographically definable components which, for descriptivepurposes, will be referred to as components A, B, C and D. Component A(a) exhibitsa moderately electron-dense fibrillogranular texture and is seen to be made up ofrather tightly arranged convoluted fibrillar elements, ranging from 6 to 10 nm inwidth, randomly intermingled with more electron-dense granules approximately15 nm in diameter. In all nucleolar profiles examined, this fibrillogranular componentoccupies most parts of the reticulated framework. Because of its abundance and ratheruniform distribution, the component in question has the appearance of a matrix inwhich the other structural components (B, C and D) of the nucleolus are embedded.Component B(i) is of low electron opacity and consists of loosely dispersed and ill-defined fibrillar elements indistinguishable from those present in the surroundingnucleoplasm. This component fills the numerous electron-transparent interstices ofthe reticulated nucleolar framework. In some nucleolar profiles, the interstices arerounded or slightly elongated, but in others they are much more irregular in shape andappear to form some sort of canalicular network. It is of interest to note also that in anumber of nucleolar profiles, structural continuity between the content of some of themore peripherally located interstices and the surrounding nucleoplasm is observed(Figs. 17, 18, arrows). Component C(c), the most electron-opaque structural elementof the nucleolar mass, is made up of closely packed convoluted fibrils, 6-10 nm indiameter, possibly with an intervening, seemingly amorphous, matrical substance.All nucleolar profiles examined show this component to be present in the peripheralportion of a number of small rounded or slightly elongated fibrillar islands quitewidely and evenly distributed within the nucleolar mass. Such fibrillar islands, witha diameter of 0-5-0-8 /im, are bordered, in part, by elements of the fibrillogranularframework with which they blend more or less imperceptibly, and, in part, by someof the electron-transparent interstices. Component D (d) is only slightly less electron-dense than component C and consists exclusively of closely arranged fibrillar elements6-10 nm in width. This component is invariably seen to occupy the more-or-lesscentral portion of the previously described fibrillar islands scattered within thenucleolar 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 materialof component C. In all nucleolar profiles examined, the small areas occupied by

642 L. A. Chouinard

component D present an outline that tends to be rounded or slightly elongated. Be-cause of their central location and fibrillar texture, these nucleolar areas containingcomponent D will be referred to as 'fibrillar centres', a term recently coined byRecher, Whitescarver & Briggs (1969) to describe comparable areas in nucleoli ofhuman tissue culture cells. Judging from their size and topographical distribution,there can be little doubt that the larger fibrillar islands just described correspond tothe more intensely stained clumps of material observed within the nucleolar mass inthe light microscope (Figs. 2-4). The nucleolus-associated chromatin (nac) appearsslightly more electron-dense than the nucleoplasm and is seen to consist of ratherloosely knit fibrillar elements 6-10 nm in width.

Visual comparison of nucleolar profiles reveals that rapid growth of the nucleolarmass during the unilaminar follicle stage is brought about mainly through an increasein the overall dimensions of the fibrillogranular framework accompanied by a parallelincrease 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 becomeresolvable in the light microscope (Fig. 4). An increase in the number and possiblyalso the size of some of the fibrillar islands with their contained fibrillar centres alsocontributes, but to a much smaller extent, to the overall growth of the nucleolar massduring the same period.

The bilaminar follicle stage. During the transition from the uni- to the bilaminarfollicle stage (postnatal days 5 and 6), most of the previously described barely resolv-able unstained spaces observed within the nucleolar mass, by light microscopy,gradually disappear and a number of small but clearly recognizable rounded un-stained areas, designated from now on as nucleolar vacuoles, make their appearancewithin 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 ofthese vacuoles, some eventually reaching up to 1-5/tm in diameter (Figs. 7, 8).Favourably transected nucleoli also reveal the existence of a barely definable core ofstainable material within these enlarging vacuoles (Figs. 7, 8, arrows). By the end ofthe bilaminar follicle stage (postnatal day 8), the nucleolus has grown into a vacuolatedbut otherwise densely and homogeneously stained spherical body which may reach upto 7 /tm in diameter (Fig. 8). There can be little doubt that the appearance and sub-sequent enlargement of the nucleolar vacuoles contribute significantly to the increasein 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-densefibrillogranular framework, (2) numerous and evenly distributed electron-transparentinterstices, of varying size and shape, filled with a material whose texture resemblesthat of the nucleoplasm, and (3) a number of small widely scattered pleomorphicislands usually made up of an outer slightly more electron-dense and an inner lesselectron-dense ('fibrillar centre') fibrillar material (Fig. 21). In the course of postnataldays 6 and 7, several profound ultrastructural and organizational changes take placemore or less concomitantly within the growing nucleolar mass (Figs. 22-24). Themain features of such intranucleolar transformations are outlined as follows: (1) Most


parts of the moderately electron-dense fibrillogranular framework gradually becomepredominantly fibrillar in texture as a result of what appears to be an unravelling orunfolding of its constituent 15-nm granules. A portion of a nucleolus undergoingsuch a degranulation process is depicted in Fig. 22. (2) As the nucleolus undergoesgradual degranulation, the outer slightly more electron-dense regions of the previouslydescribed fibrillar islands become no longer recognizable as such; the inner region('fibrillar centres') of the same islands, however, apparently persist as roundedfibrillar areas but only slightly less electron-dense than the rest of the stainable portionof the nucleolar mass (Fig. 22). (3) The light-staining interstices decrease rapidly bothin size and number as a result of what appears to be the gradual accumulation withintheir interior of a fibrillar material matching in density that present in the nucleolarframework itself; remnants of these interstices are represented by small, rounded,lightly stained spaces containing nucleoplasm-like material (Figs. 23, 24). (4) As thenucleolar mass assumes a more compact appearance, other rounded, lightly stainedspaces 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 theirbordering interstices (Figs. 23, 24). It is of interest that small rounded patches ofnucleolar material usually located adjacent to these nucleolar vacuoles retain for awhile their original fibrillogranular texture (Fig. 24). Examination of a number offavourably transected nucleoli also reveals the presence within the emerging nucleolarvacuoles of a centrally or paracentrally located core of fibrillar material of variablesize and electron density and which is thought to correspond, at least in part, to thenucleolar areas previously designated as fibrillar centres (Figs. 23, 24). The remainingportion of the nucleolar vacuoles is occupied by a fibrillar material, the texture anddensity of which resemble that of the nucleoplasm.

By postnatal day 8, the dense portion of the nucleolar mass is seen to consist, for themost part, of an intricate feltwork of convoluted fibrillar elements, 6-10 nm indiameter, lying in what appears to be a dense ill-defined amorphous matrix (Figs. 25,26). Small rounded patches of dense fibrillogranular material are still occasionallyseen located adjacent to the vacuolar space (Fig. 25). The dense portion of the nucleolarmass 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 sizeand electron opacity, within the enlarging nucleolar vacuoles (Figs. 25, 26). Theremaining portion of the vacuolar space is occupied by a material, the texture andelectron 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 observedwithin the vacuolar core itself (Figs. 25, 26). Occasionally, narrow communicationchannels between adjacent nucleolar vacuoles and between nucleolar vacuoles and thesurrounding nucleoplasm are observed.

The plurilaminar follicle stage. During transition from the bi- to the plurilaminarfollicle stage (postnatal days 9 and 10), all nucleolar profiles display, in the lightmicroscope, several rounded vacuolar areas, of varying size, and partially filled witha material which stains only slightly less intensely than the rest of the nucleolar mass

644 L. A. Chouinard

(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, someeventually reaching up to 3 /tm in diameter. As the vacuoles grow larger, they alsobecome almost completely filled with stainable material (Figs. 11, 12). During post-natal days 12 and 13, the contours of the nucleolar vacuoles in question become in-creasingly difficult to delineate because the stainability of their content comes graduallyto match that of the rest of the nucleolar mass (Figs. 13, 14). By the end of the growthperiod of the oocyte (postnatal day 14), the nucleolus has grown into a densely anduniformly stained spherical body, which may reach up to 9/tm 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 numeroustiny and evenly distributed electron-transparent areas (remnants of the nucleolarinterstices), as well as a number of much larger rounded spaces (nucleolar vacuoles)containing a nucleoplasm-like substance and variable amounts of a moderatelyelectron-dense fibrillogranular material (Figs. 27-30). The electron-dense portionof the nucleolar mass is made up of a feltwork of closely packed and randomlyoriented fibrils, 6-10 nm in diameter, apparently immersed in an amorphous matricalsubstance not readily analysed in the micrographs. The nucleolar interstices containa loosely dispersed fibrillar material matching that of the nucleoplasm in density.The moderately electron-dense material that gradually accumulates within thevacuole interior consists of intermingled masses made up of closely arranged fibrillarelements, 6-10 nm in width, interspersed with electron-dense granules of ribo-somal dimensions (Figs. 27-30). Favourable sections reveal that, in places at theperiphery of the vacuolar spaces, the contained fibrillogranular material blends more-or-less imperceptibly with the surrounding dense fibrillar material of the nucleolarmass (Figs. 27-30, arrows). The previously described intravacuolar fibrillar corebecomes no longer recognizable as such as soon as the fibrillogranular material beginsto accumulate within the interior. In the course of postnatal days 12 and 13, the lightlystained nucleolar interstices disappear gradually from view and the accumulated fibrillo-granular material of the nucleolar vacuoles becomes transformed into a compactsubstance, exclusively fibrillar in texture, and eventually matching the rest of thenucleolar mass in electron density (Fig. 31). The degranulation process of the fibrillo-granular material contained within the nucleolar vacuoles bears strong resemblanceto that which also takes place in the fibrillogranular framework of the same nucleolusduring the bilaminar follicle stage (compare Fig. 22).

By postnatal day 14, the prominent nucleolus appears as a dense compact massexhibiting no internal structural organization (Fig. 32). The fine texture of thenucleolar material, because of its compactness, is not readily analysed in ordinarypale-gold sections examined under electron microscopy. However, grey-silver sectionsprovide enough transparency to resolve the texture of this apparently homogeneousmaterial into a bewildering array of minute punctate and linear profiles of varyingsize and density (Fig. 32, inset). This characteristic appearance is probably bestinterpreted as resulting from the longitudinal and transverse sectioning of a feltworkof 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 sizerange as the thicknesses of linear profiles. In order to account for the relatively highelectron opacity of the mature oocyte nucleolus, it appears more than likely that thefeltwork of nucleolar fibrils is also permeated by some sort of amorphous matricalsubstance not readily characterized in the micrographs.

DISCUSSION

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

The oocyte nucleolus during the unilaminar follicle stage

The occurrence of a fibrillogranular framework, interstices and fibrillar islandswithin 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, althoughthe designation of these various nucleolar components has differed greatly (see Vincent& Miller, 1966; Birnstiel, 1967; Hay, 1968; Bernhard & Granboulan, 1968; Busch &Smetana, 1970). The further subdivision of the fibrillar islands of the nucleolar massinto an outer more electron-dense and an inner less electron-dense area has also beenrecorded by several authors in recent years and in a variety of cell types (Schoefl, 1964;Yasuzumi & Sugihara, 1965; Terzakis, 1965; Jezequel, Seeve & Steiner, 1967; Recheret al. 1969; Recher, Whitescarver & Briggs, 1970; Shinozuka, 1970; Simard, 1970;Hardin, Spicer & Malanos, 1970). The inner, less electron-dense areas ('fibrillarcentres' of Recher et al. 1969) of the fibrillar islands have usually been considered asa distinct structural component of the nucleolus, rich in pepsin-digestible protein andRNA; also, the possibility of DNA being present in such fibrillar centres has not beenexcluded (Recher et al. 1969, 1970).

On the basis of some of our observations, and in the light of accumulated knowledgeconcerning the spatial relationship of these various nucleolar components to thenucleolar organizing region (NOR) of the nucleolar chromosome in general (seereviews by Kopac & Mateyko, 1964; Birnstiel, 1967; Busch & Smetana, 1970), it istempting to postulate that the so-called fibrillar centres seen in all nucleolar profilesof 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 associatedmaterial belonging to the nucleolar organizing segment of the nucleolar chromosome(s).In our material, support for such a suggestion would come mainly from the followingobservations and considerations, (a) Throughout the unilaminar follicle stage, thecontent of the fibrillar centres is seen to exhibit an electron density matching quiteclosely that of the more condensed regions of the chromatin mass associated with thenucleolar surface on postnatal days 1 and 2 (Figs. 17,18). (b) Removal of intranucleolar

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DNA enzymically from formaldehyde-fixed material (Chouinard, 19706) induces anoticeable reduction in heavy metal uptake by both the fibrillar centres and thenucleolus-associated chromatin of the oocyte. (c) On postnatal days 1 and 2, favourablesections of the nucleolus reveal structural continuity between the content of some ofthe fibrillar centres and the fibrillar content of the nucleolus-associated chromatin. (d)As the nucleolus enlarges and the number of fibrillar centres per nucleolar profileincreases, the mass of nucleolus-associated chromatin decreases in size and eventuallydisappears, 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 associ-ated with the dense fibrillar component of the nucleolar mass, as seems to be the casefor intranucleolar chromatin in most other investigated types of nucleoli in eukaryoticcells, 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 growingoocyte nucleolus, during the unilaminar follicle stage, would very likely reveal that theintranucleolar interstices are, in fact, part of an intricate system of intercommunicatingchannels filled, at least in part, with nucleoplasm. Such a system undoubtedly playsan essential role in nucleolar growth by increasing the surfaces of functional exchangesand by facilitating the transport and accumulation of materials from both intra- andextranucleolar origin.

The oocyte nucleolus during the bilaminar follicle stage

Since our observations provide no evidence for the release of the granular elementsof the nucleolus into the surrounding nucleoplasm, the degranulation process of thenucleolar mass is best interpreted in terms of conversion of the 15-nm granules intofibrils through unravelling or unfolding. The observations of Marinozzi (1964) andSmetana, Unuma & Busch (1968), on the 2 basic structural components (fibrils andgranules) of the nucleolus, clearly indicate that the 15-nm granules are, indeed,wrapped-up fibrils and that transitional configuration forms frequently occur. Thereis also strong evidence that the nucleolar fibrils are the precursors of the nucleolar15-nm particles (Granboulan & Granboulan, 1965; Geuskens & Bernhard, 1966).Conversely, degranulation of the nucleolar mass through unravelling of its granularcomponents has also been produced experimentally following exposure of culturedanimal cells to supranormal temperature; such a process has also been shown to bereversible (Simard & Bernhard, 1967; Simard, 1970).

A 3-dimensional reconstruction of the oocyte nucleolus would very likely revealhere also that the nucleolar vacuoles are not isolated spaces, but part of an intra-nucleolar 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 servesprimarily as a means of increasing the surfaces of functional exchanges and facilitatingthe 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 fromthe previously described fibrillar centres, since the nucleolar vacuoles arise at someof the sites formerly occupied by the fibriUar areas and contained fibrillar centres ofthe nucleolar mass. The vacuolar cores, Like the previously discussed fibrillar centres,could then be tentatively considered as corresponding to cross- or oblique sections ofthe nucleolar organizing segment(s) of the nucleolar chromosome(s). It is conceivablealso that the vacuolar cores are but the morphological manifestation of a phenomenonrelated to rDNA amplification. Such a phenomenon has recently been shown to occurnot only in multinucleolate, but also in uninucleolate oocytes (Brown & Dawid, 1969;Dawid & Brown, 1970).

The oocyte nucleolus during the plurilaminar follicle stage

The present study being essentially morphological, no definite conclusion can bedrawn concerning the origin of the fibrillogranular material that accumulates insidethe nucleolar vacuoles. In relation to this problem, 2 possibilities, which are notmutually exclusive, should, nevertheless, be envisaged. One would be that most, if notall, of this material simply represents the accumulated products of the functionalactivity of the rest of the nucleolar mass. The frequent observation that the materiallocated in the peripheral portion of the vacuoles merges more-or-less imperceptiblywith that of the nucleolar body proper would be consistent with such a view. Most ofthe previous studies on nucleolar vacuoles in both plant and animal cells also suggestthat these structures indeed represent sites of accumulation rather than sites ofsynthesis of their contained material (Esper, 1965; Barlow, 1970). Another possibilitywould be that the nucleolar vacuoles themselves are the sites of synthesis of at leastpart of their contained material. It is conceivable, for instance, that the core of fibrillarmaterial shown to be present within such vacuoles is somehow instrumental in thesynthesis of vacuolar material. Such an interpretation would be considerablystrengthened 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 withDNA - even if the vacuoles do not contain any stainable DNA.

The fibrillar appearance of the nucleolus toward the end of mammalian oogenesishas already been noted by Parsons (1962) and Adams & Hertig (1964). In the fullygrown mammalian oocyte, the fibrillar content of the nucleolus constitutes very Likelya stable storage form for the accumulated nucleolar material (Hay, 1968).

In summary, all of our observations would be consistent with the generally heldview that the nucleolus, during growth of the primary oocyte, is the site of massivesynthesis 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 cyto-plasm only later on, at the time of nuclear envelope breakdown, and eventually utilizedduring early embryogenesis (Davidson, Crippa, Allfrey & Mirsky, 1966; Crippa,Davidson & Mirsky, 1967; Davidson, 1968).

648 L. A. Chouinard

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

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OAKBERG, E. F. (1967). H'-uridine labelling of mouse oocytes. In Proceedings of the Colloquiumon Physiology and Reproduction in Mammals, Paris, 1966, Archs Anat. microsc. Morph. exp.56, Suppl. 3-4, pp. 171-184.

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(Received 25 March 1971)


Figs. 1-16. Series of photomicrographs, taken at a uniform magnification ( x 2800),which have been arranged in a sequence to illustrate the changes undergone by thenucleolus of the growing mouse oocyte during the unilaminar (Figs. 1-4), thebilaminar (Figs. 5—8) and the plurilaminar follicle stages (Figs. 9—16).

Fig. 1. The small densely stained nucleolus (n) is quite homogeneous in internalstructure. A sizeable mass of chromatin material (nucleolus-associated chromatin,nac) is seen in intimate contact with the surface of both the nucleolus and the nuclearmembrane.

Figs. 2, 3. The nucleolus («) contains a number of small scattered clumps (arrows)of a material which stains slightly more intensely than the rest of the nucleolar mass.

Fig. 4. The nucleolus (n) exhibits a more heterogeneous appearance. In additionto small scattered clumps of more intensely stained material (arrows), the nucleolusis seen to contain numerous barely resolvable unstained spaces distributed more-or-less evenly within its mass.

Fig. 5. The nucleolus (n) appears more homogeneous in internal structure; thepreviously described more densely stained clumps of material and the unstainedspaces are difficult to distinguish from the rest of the nucleolar mass.

Fig. 6. A number of small rounded and evenly scattered unstained areas, the nucleolarvacuoles (v) are clearly visible within the otherwise densely and uniformly stainednucleolar body (n).

Fig. 7. One of the small vacuolar spaces (v) is seen to contain a barely definable core(arrow) of stainable material.

Fig. 8. Two small and one large vacuoles (v) contain a centrally or paracentrallylocated core (arrows) of stainable material.

Figs. 9-12. The nucleolar profiles (n) display several rounded vacuolar areas (v),of varying size, and partially filled with a material which stains slightly less intenselythan the rest of the nucleolar mass.

Figs. 13, 14. The stainability of the content of the vacuoles (u) matches quite closelythat of the rest of the nucleolar mass (n).

Figs. 15, 16. The nucleolus (n) appears as a densely and uniformly stained sphericalbody.

Figs. 17-20. Electron micrographs depicting the ultrastructural organization of themouse oocyte nucleolus during the unilaminar follicle stage (Fig. 17, postnatal day 1,x 26000; Fig. 18, postnatal day 2, x 26000; Fig. 19, postnatal day 3, x 23000; Fig. 20,postnatal day 4, x 28000). Throughout that stage, the nucleolus shows an overallreticulated-type of structure, consisting of 4 components referred to as A, B, C and D.Component A (a) exhibits a moderately electron-dense fibrillogranular texture and isseen to occupy most parts of the reticulated framework. Component B(6) is of lowelectron opacity and consists of a nucleoplasm-like fibrillar material filling thenumerous irregularly shaped interstices of the nucleolar framework. In places (Figs.17, 18, arrows), communication between the nucleoplasm and the interstices inquestion is observed. Component C(c), the most electron-opaque structural elementof the nucleolar mass is fibrillar and occupies the peripheral portion of a number ofsmall rounded or slightly elongated fibrillar islands quite widely and uniformly scatteredwithin the nucleolar body. Component D (d), also fibrillar in texture and only slightlyless electron-dense than component C, occupies the more-or-less central portion ofthe previously described fibrillar islands. In the course of postnatal days 1 and 2 (Figs.17, 18) a mass of chromatin material (nucleolus-associated chromatin, nac) is seen inintimate contact with the surface of both the nucleolus and the nuclear membrane(nm).

42 C E L 9

652 L. A. Chouinard

10*For legend see p. 651.

Nucleolus in growing mouse oocyte &S3

n.*>

11 '

• -13

i

14

n

For legend see p. 651.

654 L. A. Chouinard

18


656 L. A. Chouinard

Figs. 21-26. Electron micrographs depicting some of the ultrastructural andorganizational changes undergone by the mouse oocyte nucleolus during the bilaminarfollicle stage (Fig. 21, postnatal day 5, x 28000; Fig. 22, postnatal day 6, x46000;Figs. 23, 24, postnatal day 7, x 32000; Figs. 25, 26, postnatal day 8, x 32000).

Fig. 21. The nucleolus exhibits an overall reticulated type of structure consisting of4 ultrastructurally definable components: (1) a moderately electron-dense fibrillo-granular framework (component A); (2) numerous and evenly distributed electron-transparent interstices of varying size and shape filled with nucleoplasm-like material(component B); (3) a number of widely scattered pleomorphic islands, usually madeup of an outer more electron-dense material (component C) and (4) an inner lesselectron-dense fibrillar material (component D).

Fig. 22. Portion of a nucleolus in the process of undergoing degranulation of itsfibrillogranular framework. In places, this nucleolar framework still consists of afibrillogranular material (Jg) made up of closely arranged convoluted fibrils 6-10 nmin width, interspersed with granules of ribosomal dimensions; in other places, thenucleolar framework is made up predominantly of fibrillar material (/). The materialcontained within the nucleolar interstices (i) exhibits a texture and electron densitymatching those of the surrounding nucleoplasm. The small rounded fibrillar area(circled by arrows) of slightly lower electron opacity than that of the rest of thenucleolar framework is thought to correspond to a fibrillar centre.

Nucleolus in growing mouse oocyte

\

657

658 L. A. Chouinard

Figs. 23-26. The dense portion of the nucleolar mass, consisting predominantly of anintricate fibrillar feltwork embedded in a dense ill-defined amorphous matrix, ispermeated by a number of tiny electron-transparent spaces, remnants of the nucleolarinterstices (t), and by much larger lightly stained spaces corresponding to the enlargingnucleolar vacuoles (v). The vacuoles contain a centrally or paracentrally located core(circled by arrows) of fibrillar material of variable size and electron density; theremaining portion of the vacuolar space is occupied by a fibrillar material, the textureand density of which resemble that of the nucleoplasm and also in Figs. 25, 26, by anumber of tiny scattered and ill-defined clumps of fibrillar material. In Figs. 24 and 25,round patches of fibrillogranular material (Jg) are seen adjacent to the nucleolarvacuoles.

b

f

Nucleolus in growing mouse oocyte

\

\

660 L. A. Chouinard

Figs. 27-32. Electron micrographs depicting some of the ultrastructural and organ-izational changes undergone by the mouse oocyte nucleolus during the plurilaminarfollicle stage (Figs. 27-30, postnatal days 9-12, x 32000; Fig. 31, postnatal day 13,x 36000; Fig. 32, postnatal day 14, x 36000; Fig. 32, inset, x 38000).

Figs. 27-30. The dense portion of the nucleolus is made up of a feltwork of closelypacked and randomly oriented fibrils, 6—10 nm in diameter, apparently immersed inan amorphous matrical substance not readily analysed in the micrographs. Thenucleolar interstices (1) contain a loosely dispersed fibrillar material matching thatof the nucleoplasm in electron density. The moderately electron-dense materialpresent within the vacuole (v) interior consists of intermingled masses made up ofclosely arranged fibrillar elements, 6—10 nm in width, interspersed with electron-dense granules of ribosomal dimensions. In places (arrows) at the periphery of thevacuolar spaces, the contained fibrillogranular material is seen to blend more or lessimperceptibly with the surrounding dense fibrillar material of the nucleolar mass.

662 L. A. Chouinard

Fig. 31. The bulk of the nucleolar mass consists of a feltwork of densely packed andrandomly oriented fibrils, 6-10 nm in width, associated with a seemingly amorphousmatrical substance. One medium-sized vacuole (v) and a grazing section of another,as well as a few remnants of the nucleolar interstices (i) are clearly recognizable in theperipheral portion of the nucleolar mass. The larger vacuole is filled to a considerableextent with a fibrillogranular material in process of undergoing gradual degranulation.

Fig. 32. The nucleolus appears as an electron-dense compact mass exhibiting nointernal structure. The fine texture of the nucleolar material, because of its compact-ness, is not readily analysed in ordinary pale-gold sections examined at this lowmagnification. Grey-silver sections, however, provide enough transparency to resolvethe texture of this apparently homogeneous material into an array of minute punctateand linear profiles of varying size and density (inset). This characteristic appearanceis probably best interpreted as resulting from the longitudinal and transverse sectioningof a feltwork of randomly oriented fibrils 6—10 nm in diameter. The feltwork of fibrilsalso appears to be associated with some sort of amorphous matrical substance.

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