A LIGHT- AND ELECTRON-MICROSCOPE STUDY OF THE OOCYTE NUCLEUS DURING DEVELOPMENT OF THE ANTRAL FOLLICLE IN THE PREPUBERTAL MOUSE

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1 J. Cell Sci. 17, (197s) 589 Printed in Great Britain A LIGHT- AND ELECTRON-MICROSCOPE STUDY OF THE OOCYTE NUCLEUS DURING DEVELOPMENT OF THE ANTRAL FOLLICLE 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 mouse oocyte nucleus during the preparatory, the maturative and the preovulatory stages of antral follicle development, have been studied under both light and electron microscopy. All observations have been made on those antral follicles whose development is initiated on postnatal day 14 and completed by postnatal day 28 in prepubertal animals of the ICR albino mouse strain. The formed entities that can be recognized within the oocyte nucleus during that period are the condensing bivalents, the heterochromatic knobs, the nucleolus and the extranucleolar bodies. At the onset of antral follicle development, the highly unravelled dictyate bivalents are seen to take on a lampbrush-type configuration. Subsequent condensation of these lampbrush bivalents appears to be a very gradual and lengthy process that extends over almost the entire period of antral follicle development. The shortening and thickening of the lampbrush bivalents are best interpreted as resulting from the withdrawal of their lateral loop-like projections into the chromosome axes and from the focal aggregation of these axes into compact chromatin masses. Electron-opaque granules, which appear within the oocyte nucleus during the preparatory and maturative follicle stages, are seen to be intimately associated with these condensing bivalents. A number of Feulgen-positive heterochromatic knobs make their appearance in contact with certain bivalents during the preparatory follicle stage. These knobs are not reincorporated as such into the condensing chromatin masses and undergo disintegration and dissolution during the preovulatory follicle stage. The size, shape and ultrastructural features of the nucleolus remain unchanged throughout the period of antral follicle development. Breakdown and dissolution of the nucleolar mass is a swift process that takes place only in the fully mature preovulatory follicle and more or less concomitantly with the dismantling of the nuclear envelope. The extranucleolar bodies increase noticeably in size during the preparatory and the maturative follicle stages; they shrink in size and undergo dissolution during the preovulatory stage of antral follicle development. An attempt is made to interpret these morphological changes in the light of current knowledge concerning the architectural and functional organization of the oocyte nucleus in general during meiotic prophase. The relevant observational evidence would be consistent with the view that, during antral follicle development, the mouse oocyte nucleus is not, as too often assumed, in a period of arrested evolution; its formed components undergo structural, maturational and functional changes which are of significance not only for the resumption of the first meiotic prophase but also for the early development of the embryo. INTRODUCTION As is well known, the development of a mammalian ovarian follicle with its contained oocyte takes place in 2 phases. The first phase is primarily one of growth of the oocyte itself which occurs during the transformation of a unilaminar into a 38 CEL 17

2 59 L. A, Chouinard plurilaminar follicle with first appearance of the antrum. The second is primarily one of growth of the antral follicle itself due to proliferation of the follicular cells and expansion of the antrum; during that phase, the contained oocyte shows no appreciable enlargement (Franchi, Mandl & Zuckerman, 1962; Wischnitzer, 1966; Mauleon, 1967; Peters, 1969). Previous observations have provided a descriptive account of the structural changes which occur in the nucleus during the growth phase of the primary oocyte in the prepubertal mouse (Chouinard, 1971, 1973). It is the purpose of this paper to analyse, under both light and electron microscopy, the sequence of morphological transformations undergone by the nuclear structures of the oocyte during development of the antral follicle, also in the prepubertal mouse. To the author's knowledge, this problem has received only very limited attention in the past (Mandl, 1963). However, details concerning the fine structure of the mouse oocyte nucleus in mature preovulatory (Graafian) follicles and, more specifically, at the time of nuclear envelope breakdown, have in recent years been provided by Merchant & Chang, 1971; Calarco, Donahue & Szollosi, 1972; Calarco, 1972; Szollosi, Calarco & Donahue, 1972; Zamboni, MATERIALS AND METHODS 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, development of a number of antral follicles is initiated on or around postnatal day 14 and completed by postnatal day 28. In order to secure all development stages of the antral follicle in question - in principle, the largest antral follicle to be observed in any given ovary at any given time postpartum - neonatal mice were sacrificed at daily intervals from postnatal day 14 up to postnatal day 28. All of our observations have been made on the oocytes contained within such developing antral follicles. It should be stressed at this point that, at each postnatal day, the antral follicles selected for study were among the largest present in the ovary and that none including their contained oocytes show any visible sign of involution or atresia. Thus, it can be confidently assumed that these antral follicles were, at the time of fixation, in a normal and active state of differentiation and development. The techniques employed for the study of the mouse oocyte nucleus by both light and electron microscopy were described previously (Chouinard, 1971). OBSERVATIONS Development of the antral follicle For descriptive purposes, the development period of the antral follicles studied - i.e. those in which development is initiated on or around postnatal day 14 and completed by postnatal day 28 - will be divided into 3 successive stages depending on the extent of follicle development, and these will be referred to as the preparatory, the maturative and the preovulatory follicle stages. It should be borne in mind that these stages, though fairly distinct, are, nevertheless, only periods in a continuous process of follicular development and therefore connected with each other by intermediate conditions. The preparatory follicle stage. On postnatal day 14, the larger follicles, which may reach up to 250/tm in diameter, are seen to contain a narrow eccentric and usually crescent-shaped antrum containing liquor folliculi. The oocyte, which has already

3 Oocyte nucleus in developing antral follicle 591 reached its maximum diameter (70 /tm) occupies the central region of the follicle and is surrounded by cells of the cumulus oophorus; the cells at the base of the cumulus are attached over a wide area to those of the multilayered stratum granulosum. During the preparatory follicle stage (postnatal day 14-20), the antral follicle undergoes a gradual increase in volume as a result of: the intensive proliferative activity of the cells in both the cumulus oophorus and the stratum granulosum; and the continued accumulation of liquor folliculi into the widening antrum. By postnatal day 20, the larger antral follicles, which measure up to 350 /im in diameter, exhibit an expanded eccentrically located crescent-shaped antrum. The oocyte, which continues to assume a central position, is encompassed by 5-6 layers of follicular cells in the cumulus. The maturative follicle stage. During that stage which extends from postnatal day 20 to approximately postnatal day 26, the antral follicle continues to enlarge progressively until it is approximately 450 /tm in diameter. Such an enlargement is brought about partly by an increase through mitosis in the number of cells of both the stratum granulosum and cumulus oophorus, and partly by an increase in the amount of liquor folliculi contained within the expanding antrum. By postnatal day 26, the bulk of the follicle comes to be composed of a large central rounded pool of liquor folliculi surrounded by cells of the stratum granulosum. The eccentrically located oocyte, together with the cells that cover it, are still broadly connected, on one side, to the stratum granulosum and project into the antral cavity. By the end of the maturative follicle stage (postnatal day 26), typical antral follicular organization can therefore be said to be completed. The preovulatory follicle stage. In the course of postnatal days 27 and 28, the antral follicles, destined to ovulate at first oestrous, undergo a final spurt of preovulatory growth. The increase in follicular cells population is, together with the accompanying secretion of liquor folliculi, the important factor in follicular expansion during that period. Due to this expansion, the antral follicle extends to the surface of the ovary and eventually bulges out into the periovarian cavity. Concomitantly, small liquidfilled cavities (secondary liquor folliculi) appear among the cells of the cumulus, thus causing a gradual detachment of the primary oocyte together with its invested cells (corona radiata) from the underlying stratum granulosum. In nearly matured antral follicles, the primary oocyte encompassed by 5-6 layers of coronal cells usually becomes free-floating in the follicular fluid of the antrum. By the end of our observation period (postnatal day 28), the larger antral follicles, which may reach up to 550/mi in diameter, have thus acquired the classical preovulatory characteristics. Ovulation occurs soon after these conditions are established. The nuclear structures of the oocyte during development of the antral follicle During development of the antral follicle, the mouse oocyte (70 /tm in diameter) and its contained nucleus (22-24/im ' n diameter) show no appreciable increase in size. During the same period, however, the structural components of the oocyte nucleus (bivalents and associated structures) undergo gradual configurational and 38-3

4 592 L. A. Chouinard dimensional changes. For the sake of description, the sequence of such changes will be defined in relation to the 3 stages of antral follicle development described in the previous section, namely the preparatory, the maturative and the preovulatory follicle stages. The preparatory follicle stage. At the onset of antral follicle development (postnatal day 14), 2 types of toluidine blue-stained and Feulgen-negative structural components are clearly recognizable within the oocyte nucleus (Fig. 1). The first type corresponds to the prominent nucleolus, which appears as a densely and uniformly stained spherical mass, approximately 9 /tm in diameter, located more or less centrally within the nucleus. The second type is represented by several moderately stained rounded masses, / fm m diameter. Such masses, which have previously been referred to as extranucleolar bodies (cf. Chouinard, 1973), usually lie widely separated from one another and at varying distances from the nucleolus and the undulating nuclear envelope. Up to four extranucleolar bodies have been observed in a single nuclear profile, thus indicating that their number per nucleus is relatively high. During the preparatory follicle stage, the nucleolus shows no detectable change either in size, shape, location or staining behaviour (Figs. 1 6); the extranucleolar bodies, on the other hand, undergo during the period extending from postnatal day 16 to 20 a noticeable increase in size, some eventually reaching up to 3-5 /mi in diameter (Figs. 4-6). On postnatal day 14, the dictyate bivalents are still in a very extended diffuse state and, therefore, not recognizable as such within the nucleus (Fig. 1). In the course of postnatal day 15 to 16, the dictyate bivalents seemingly undergo a change in configuration and appear in the form of a faintly stained network of convoluted chromatin threads quite uniformly distributed within the nuclear cavity (Figs. 2, 3). As growth of the antral follicle proceeds (postnatal day 16-20), the loosely organized bivalents eventually reach a slightly more advanced state of condensation and, in places, are seen to take on a configuration somewhat reminiscent of that of the socalled lampbrush chromosomes (Figs. 4-6). More or less concomitantly, a number of barely resolvable rounded Feulgen-positive, toluidine blue-stained heterochromatic knobs become recognizable within the nuclear cavity. Favourable sections reveal that such Feulgenpositive heterochromatic knobs lie in close association with segments of the early condensing bivalents and also with the surface of the previously described extranucleolar bodies. In the course of postnatal days 18-20, some of these chromatin knobs and, specially those observed in contact with the surface of extranucleolar bodies, undergo a noticeable increase in size, some eventually reaching up to 1-5 fim in diameter (Figs. 4-6). At the onset of the preparatory follicle stage, the formed entities of the oocyte nucleus are seen to exhibit the following distinctive ultrastructural features. The prominent nucleolus appears as a dense compact mass showing no internal structural organization (Fig. 19). 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. 19, inset). This charac-

5 Oocyte nucleus in developing antral follicle 593 teristic appearance is probably best interpreted as resulting from the longitudinal and transverse sectioning of a feltvvork of tightly packed randomly oriented fibrils which are 6-10 nra in diameter. The punctate profiles would then represent fibrils seen in transverse sections, since they have the same size range and the thicknesses of linear profiles. In order to account for the relatively high electron opacity of the nucleolus, it appears more than likely that the feltwork of nucleolar fibrils is also permeated by some sort of amorphous matrix not readily characterized in the micrographs. The extranucleolar bodies, on the other hand, exhibit a fibrillogranular texture; they each consist of intermingled masses made up of rather closely arranged convoluted fibrils, 6 10 nm in width, in which are uniformly interspersed moderately electron-dense granules approximately 15 nm in diameter; these constituent elements appear to be embedded in some sort of ill-defined substance ultrastructurally similar to that of the surrounding nucleoplasm (Fig. 20). During the preparatory follicle stage, the nucleolus shows no detectable change in its staining behaviour and ultrastructural characteristics. The same holds true for the extranucleolar bodies even if they undergo, during that period, a noticeable increase in size (Fig. 25). On postnatal day 14 or 15, the extended bivalents are in a diffuse configurational state resulting very likely from an unravelling of both the chromosomal axes and their lateral projections throughout the nuclear cavity; in places, segments of these extended bivalents appear in the form of small, ill-defined moderately electron-dense aggregates of convoluted and loosely arranged chromatin fibrils 6-10 nm in diameter (Fig. 19). As the preparatory follicle stage proceeds, the aggregates of chromatin fibrils become somewhat coarser and slightly more electron-dense (Figs. 21, 22). Throughout the preparatory follicle stage, isolated electron-opaque granules, nm in diameter, are seen, in places, to be closely associated with the chromatin fibrils (Figs. 19, 21, 22). Occupying the remainder of the nucleoplasm is a continuous phase of low density containing a poorly defined sparse flocculent material, the nuclear sap. Under electron microscopy, the previously described heterochromatic knobs of all sizes, observed within the oocyte nucleus during the preparatory follicle stage, appear to be made up of an entanglement of closely arranged bundles of electron-dense fibrils, 8-10 nm in diameter, embedded in an ill-defined matrix of only slightly lower opacity and not readily characterized in the micrographs (Figs ). The maturative follicle stage. During that stage of antral follicle development (postnatal days 20-26), the nucleolus shows, under light microscopy, no detectable change either in size, shape, location or staining properties. Meanwhile, the extranucleolar bodies undergo a noticeable increase in size, some eventually reaching up to 4-5 /«n in diameter (Figs. 7-12). During the same period, the lampbrush bivalents undergo a still further degree of condensation and eventually stand out in much sharper contrast to the almost unstained nucleoplasmic background (Figs. 7-12). By postnatal day 26, each of the condensing lampbrush bivalents indeed appears in the form of a complex network of convoluted chromosomal threads varying from a loose arrangement in some places to a denser, more closely packed one in others. These morecondensed portions of the bivalents are seen as rounded or irregularly shaped clumps of Feulgen-positive, toluidine blue-stained material (Figs. 7-12); such clumps of

6 594 L. A. Chouinard aggregated chromatin usually stain slightly less intensely than the previously described heterochromatic knobs which continue to lie in close association with segments of the condensing bivalents and also with the surface of the extranucleolar bodies (Figs. 7-12). During the maturative follicle stage, no detectable changes can be observed in the tinctorial and ultrastructural features of the oocyte nucleolus and extranucleolar bodies (Figs. 26, 28, 29); the same holds true for the heterochromatic knobs (Figs. 27, 28) except for the occasional appearance, on postnatal days 25 and 26, of one and sometimes two rounded lightly staining areas within their mass (Fig. 29). By postnatal day 26, the less-condensed segments of the oocyte bivalent appear, under electron microscopy, in the form of irregularly shaped areas made up of loosely arranged entanglements of moderately electron-dense chromatin fibrils; isolated or small berrylike aggregates of electron-opaque granules, nm m diameter, are also seen here and there within these areas of less-condensed chromatin (Figs ). The more condensed portions of the oocyte bivalents, on the other hand, are represented by irregularly shaped masses made up of a number of small clumps of condensing chromatin material intermingled with rounded or angular clusters of electron-opaque granules, some of which may reach up to 90 nm in diameter (Figs ). The preovulatory follicle stage. During that final stage of antral follicle development (postnatal days 27 and 28), several more or less concomitant events are seen to take place within the oocyte nucleus prior to the eventual process of nuclear envelope breakdown and resumption of the first meiotic prophase (diakinesis and prometaphase I). The salient features of these nuclear events, as seen under light microscopy, are summarized in what follows and some are illustrated in Figs Firstly, the bivalents gradually enter their final phase of condensation, although apparently not at the same rate simultaneously throughout the oocyte nucleus. At the end of our observation period, indeed, the chromatin of some of the bivalents is already seen to be focally aggregated into relatively compact masses of irregular size and configuration, while the chromatin of other bivalents within the same nucleus still appears to be in a relatively uncondensed condition. The bivalents also appear to condense in contact with the undulating nuclear envelope. Secondly, the nucleolus assumes a more peripheral position within the nuclear cavity and becomes surrounded by an irregularly shaped halo of moderately condensed chromatin material; the size, shape and staining behaviour of the nucleolar mass itself, however, still remain unchanged. Thirdly, the extranucleolar bodies eventually detach from their associated bivalents and become apparently free floating within the nucleoplasm. Following detachment, the extranucleolar bodies undergo a gradual shrinkage in size until only small rounded vestiges remain; in turn, these remnants of the extranucleolar bodies disappear from view. The extranucleolar bodies in question do not appear to undergo detachment and dissolution at the same rate simultaneously throughout the oocyte nucleus; some, for instance, were still intimately associated with bivalents and of relatively large size even at the end of our observation period. Fourthly, the dense heterochromatic knobs, whether associated with the extranucleolar bodies or not, are eventually released from the condensing bivalents, undergo dissolution or disintegra-

7 Oocyte nucleus in developing antral follicle 595 tion, usually by forming a series of smaller Feulgen-positive bodies, and, finally, vanish from sight. At the ultrastructural level, the more condensed portions of the bivalents appear as agglomerations of closely packed, moderately electron-dense chromatin fibrils; the peripheral regions of these agglomerations are seen to consist of more loosely arranged chromatin fibrils in which are embedded a few usually widely scattered isolated or small clusters of electron-opaque granules (Figs. 34, 35). In the course of postnatal days 27 and 28, the oocyte nucleolus exhibits ultrastructural features indistinguishable from those already described for that nuclear organelle during the preceding stages of antral follicle development (Fig. 38, and inset). The irregularly shaped halo surrounding the nucleolar body is seen to be made up of a complex meshwork of chromatin fibrils varying from a loose arrangement in some places to a denser, more closely packed one in others; electron-opaque granules, occurring either singly or in clusters, are also observed here and there within this chromatin halo (Fig. 38). As they gradually undergo shrinkage in size, the extranucleolar bodies are still seen to consist of a loose meshwork of convoluted fibrils, 6-10 nm in width, in which are uniformly interspersed moderately electron-dense 15-nm granules (Figs. 39, 41, 42). At the boundary of such extranucleolar bodies, the constituent fibrillogranular material is seen to merge more or less imperceptibly with the surrounding loosely dispersed fibrillar components of the nucleoplasm. Figs. 36 and 37 show a dense heterochromatic knob still associated, on one side, with an extranucleolar body (Fig. 36), and, on the other side, through what appears to be a narrow chromatin stalk, with the condensed portion of a bivalent (Fig. 37). Heterochromatic knobs presumably just released from the condensing bivalents are depicted in Figs. 39 and 40; the one shown in Fig. 39 lies in close association with the surface of an extranucleolar body. Figs. 41 and 42 illustrate the process of gradual disintegration through fragmentation, of a heterochromatic knob over the surface of a shrinking extranucleolar body. Except for the occasional presence of a few small staining spaces within their mass, the newly released heterochromatic knobs appear as a tight electron-dense mass of intertwined bundles of fibrils (Figs. 36, 37, 39, 40). The fragments originating from the disintegration of the heterochromatic knobs are also seen to exhibit a compact electron-dense fibrillar texture (Figs. 41, 42). DISCUSSION Our observations reveal that the oocyte bivalents and associated structures undergo a precise pattern of morphological changes that can be correlated with development of the antral follicle in the prepubertal mouse. In the following discussion, an attempt will be made to interpret these changes in the light of current knowledge concerning the architectural and functional organization of the oocyte nucleus in general. The oocyte bivalents during development of the antral follicle At the onset of the preparatory stage of antral follicle development, the mouse oocyte bivalents are still in a highly diffuse dictyate state resulting very likely from an

8 596 L. A. Chouinard unravelling of both the chromosomal axes and their lateral projections throughout the nuclear cavity (Franchi & Mandl, 1962; Tsuda, 1965; Baker & Franchi, 1967; Chouinard, 1973). In the course of the preparatory follicle stage, the same bivalents lose their highly unravelled condition and assume a more compacted configuration somewhat reminiscent of that of the lampbrush chromosomes. Subsequent condensation of these lampbrush bivalents, in those fully grown oocytes destined to be ovulated at first oestrous, appears to be a very gradual and lengthy process that extends over almost the entire period of antral follicle development (i.e. approximately 14 days in duration). During that period, the relevant observational evidence would be consistent with the view that the gradual shortening and thickening of the lampbrush bivalents are basically the result of withdrawal of their lateral loop-like projections into the chromosomes axes and of the more or less concomitant focal aggregation of the same chromosome axes into compact chromatin masses. As is well known, both the dictyate and the lampbrush chromosomes, in their extended state, rapidly incorporate radioactive precursors of RNA and are thus considered to be the sites of intense transcriptive activity during oocyte growth (Gall & Callan, 1962; Izawa, Allfrey & Mirsky, 1963; Davidson, Crippa, Allfrey & Mirsky, 1966; Callan, 1969; Oakberg, 1967, 1968; Baker, Beaumont & Franchi, 1969; Miller, Beatty & Hamkalo, 1972). During the preparatory and the maturative stages of antral follicle development, the lampbrush bivalents of the mouse oocyte are still in a relatively expanded condition, thus raising the possibility that at least some of their lateral loops continue to be active in the formation, stabilization and packaging of gene products. Morphological expression of such functional activities during that period would be provided, in part, by the appearance within the oocyte nucleus of a large number of electron-opaque granules, and, in part, by the noticeable increase in size of the chromosome-associated extranucleolar bodies (vide infra). Support for the above interpretation comes from recent radioautographic observations of Moore, Lintern-Moore, Peters & Faber (1974) indicating that the mouse oocyte nucleus in the antral follicle continues, although at a reduced rate, to remain active in the synthesis of RNA. The present study being essentially morphological in character, no definite conclusion can be drawn concerning the functional significance of the electronopaque granules that accumulate inside the mouse oocyte nucleus during the preparatory and maturative stages of antral follicle development. In relation to this problem, it is of interest to note that such granules are invariably seen to be intimately associated with the constituent elements of the condensing bivalents, and are almost absent from those segments of the bivalents which have undergone condensation. From observations such as these, it might not be unreasonable to postulate that the electronopaque granules in question are somehow involved in the complex process of focal condensation of the oocyte bivalents during antral follicle development. During the preovulatory stage of antral follicle development, the various bivalents do not appear to undergo condensation at the same rate synchronously throughout the oocyte nucleus. For instance, the bivalents or portions of bivalents forming a halo around the nucleolar mass are seen to be in a relatively extended form even at the end of our observation period, which is probably just a few hours prior to nuclear envelope

9 Oocyte nucleus in developing antral follicle 597 breakdown and resumption of the first meiotic prophase. The presence of this extended form of chromatin, assuming that it is capable of transcriptive activities, might perhaps account for the burst in RNA synthesis which has been reported during the maturation period of the mouse oocyte just prior to nuclear envelope breakdown (Bloom & Mukherjee, 1972). The oocyte heterochromatic knobs during development of the antral follicle On the basis of what is known concerning the architectural organization of the lampbrush chromosome in general (cf. Callan, 1963, 1969), one is tempted to postulate that the dense Feulgen-positive heterochromatic knobs, which arise in close association with the oocyte bivalents during the preparatory follicle stage of antral follicle development, are - like the lampbrush chromomeres - derived from the rewinding of specially large chromatin loops involved in specific gene activities such as, for instance, the formation of extranucleolar bodies (vide infra). However, the mouse oocyte heterochromatic knobs are much larger than ordinary chromomeres and, furthermore, they are eventually released from the condensing bivalents and undergo disintegration into the nucleoplasm during the preovulatory stage of antral follicle development. The appearance and subsequent disappearance of large Feulgenpositive bodies during oogenesis has been reported several times in the past (cf. Pelc, 1972). Our observations are possibly best interpreted in the light of recent findings concerning the structural and molecular organization of the socalled major chromomeres (or DNA bodies) seen in oocytes of Acheta domesticus (Lima-de-Faria, Daskaloff & Enell, 1973; Lima-de-Faria, Gustafsson & Jaworska, 1973; Lima-de-Faria, Jaworska & Daskaloff, 1973). These authors have shown conclusively that such major chromomeres contain not only spiralized loop regions of the lampbrush chromosomes but also a large amount of amplified DNA copies. These amplified DNA copies are released into the nucleoplasm at given times of development. A similar situation may exist in the case of the heterochromatic knobs ('giant chromomeres') seen in the mouse oocyte nucleus during antral follicle development. Redundant copies of a given DNA sequence as well as amplified DNA copies of well defined genes would thus be an integral part of the structural and molecular organization of the heterochromatic knobs in question. In the preovulatory stage of antral follicle development, the redundant DNA sequence would be reincorporated into the condensing bivalent but the amplified DNA copies would be released into the oocyte nucleoplasm. Assuming the validity of the above line of reasoning, it would be of interest from a genetic and developmental point of view to know whether or not the released amplified DNA copies from the heterochromatic knobs are eventually transcribed in the cytoplasm in connexion with the information needed for the early embryonic development. The oocyte nucleolus and extranucleolar bodies during development of the antral follicle Previous findings have shown that development of the mouse oocyte nucleolus takes place during the growth period of the oocyte in pre-antral follicles (Chouinard, 1971). The rounded nucleolus of the fully grown oocyte appears as a dense mass, exclusively fibrillar in texture and exhibiting no internal structural organization. The

10 598 L. A. Chouinard present observations reveal that the size, shape and ultrastructural features of the mouse oocyte nucleolar mass remain unchanged during the entire period of antral follicle development. Thus, it can be quite safely concluded that the mouse oocyte nucleolus is in a quiescent or resting state during that period. As already reported by several authors, breakdown of the mouse oocyte nucleolus is a swift process that takes place only in fully mature preovulatory follicles and more or less concomitantly with the breakdown of the nuclear envelope and resumption of the first meiotic prophase (Donahue, 1968; Merchant & Chang, 1971; Calarco et al. 1972; Sorensen, 1973). The large amount of rrna and other functionally related non-rna material stored in the dormant nucleolus is then released into the nucleoplasm and eventually into the cytoplasm of the mature oocyte. The observations presented in a previous paper have been shown to be consistent with the view that the fibrillogranular extranucleolar bodies are morphological expression - like the puffs of the polytene and the loops or spheroids of the lampbrush chromosomes - of differential gene activity on the part of localized regions of the dictyate chromosomes during growth of the mouse oocyte (Chouinard, 1973). The present observations indicate that the fibrillogranular extranucleolar bodies in question not only persist but also increase noticeably in size during the preparatory and the maturative follicle stages of antral follicle development. Such findings are taken to indicate that genomic sites, other than those associated with the nucleolus, remain capable of synthesis and accretion of gene products even after the oocyte has reached its full size and is contained in developing antral follicles. During the preovulatory follicle stage of antral follicle development, the extranucleolar bodies detach from the condensing bivalents, shrink rapidly in size and eventually disappear from view. Following dissolution of these extranucleolar bodies, the oocyte nucleoplasm thus becomes richly provided with gene products - distinct from those originating from the nucleolar mass - which, at the time of nuclear envelope breakdown, will be released to the cytoplasm. In relation to this problem, it should be recalled that, according to current thinking, the early development of the embryo is mainly directed by gene products which have been presynthesized and stored during the protracted dictyate or diplotene stage of the first meiotic prophase (Davidson et al. 1966; Crippa, Davidson & Mirsky, 1967; Davidson, 1968; Miller et al. 1972). In summary, the relevant observational evidence obtained in the present study would be consistent with the view that, during development of the antral follicle, the mouse oocyte nucleus is not, as too often assumed, in a period of arrested evolution: its formed components undergo structural, maturational and functional changes which are of significance not only for the resumption of the first meiotic prophase but also for the early development of the embryo. This work was supported by a grant (MA-770) from the Medical Research Council of Canada.

11 Oocyte nucleus in developing antral follicle 599 REFERENCES BAKER, T. G. & FRANCHI, L. L. (1967). The structure of the chromosomes in human primordial oocytes. Chromosonia 22, BAKER, T. G., BEAUMONT, H. M. & FRANCHI, L. L. (1969). The uptake of tritiated uridine and phenylalanine by the ovaries of rats and monkeys. J. Cell Sci. 4, BLOOM, A. M. & MUKHERJEE, B. B. (1972). RNA synthesis in maturing mouse oocytes. Expl Cell Res. 74, CALARCO, P. G. (1972). The kinetochore in oocyte maturation. In Oogenesis (ed. J. D. Biggers & A. E. Schuetz), pp Baltimore: University Park Press. CALARCO, P. G., DONAHUE, R. P. & SZOLLOSI, D. (1972). Germinal vesicle breakdown in the mouse oocyte. J. Cell Sci. 10, CALLAN, H. G. (1963). The nature of lampbrush chromosomes. Int. Rev. Cytol. 15, CALLAN, H. G. (1969). Biochemical activities of chromosomes during the prophase of meiosis. In Handbook of Molecular Cytology (ed. A. Lima-de-Faria), pp Amsterdam: North-Holland Publishing. CHOUINARD, L. A. (1971). A light- and electron-microscope study of the nucleolus during growth of the oocyte in the prepubertal mouse, jf. Cell Sci. 9, CHOUINARD, L. A. (1973). An electron-microscope study of the extranucleolar bodies during growth of the oocyte in the prepubertal mouse. J. Cell Sci. 12, CRIPPA, M., DAVIDSON, E. H. & MIRSKY, A. E. (1967). Persistence in early amphibian embryos of informational RNA's from the lampbrush chromosome stage of oogenesis. Proc. natn. Acad. Sci. U.S.A. 47, DAVIDSON, E. H. (1968). Gene Activity in Early Development. New York and London: Academic Press. DAVIDSON, E. H., CRIPPA, M., ALLFRHY, V. G. & MIRSKY, A. E. (1966). Genomic function during the lampbrush stage of amphibian oogenesis. Proc. natn. Acad. Sci. U.S.A. 56, DONAHUE, R. P. (1968). Maturation of the mouse oocyte in vitro. I. Sequence and timing of nuclear progression. J. exp. Zool. 169, FRANCHI, L. L. & MANDL, A. M. (1962). The ultrastructure of oogonia and oocytes in the foetal and neonatal rat. Proc. R. Soc. B 157, FRANCHI, L. L., MANDL, A. M. & ZUCKERMAN, S. (1962). The development of the ovary and the process of oogenesis. In The Ovary, vol. 1 (ed. S. Zuckerman, A. M. Mandl & P. Eckstein), pp New York and London: Academic Press. GALL, J. G. & CALLAN, H. G. (1962). Uridine-H 3 incorporation in lampbrush chromosomes. Proc. natn. Acad. Sci. U.S.A. 48, IZAWA, M., ALLFREY, V. G. & MIRSKY, A. E. (1963). Composition of the nucleus and chromosomes in the lampbrush stage of the oocyte. Proc. natn. Acad. Sci. U.S.A. 50, LIMA-DE-FARIA, A., DASKALOFF, R. & ENELL, A. (1973). Amplification of ribosomal DNA in Acheta. I. The number of chromomeres involved in the amplification process. Hereditas 73, LIMA-DE-FARIA, A., GUSTAFSSON, T. & JAWORSKA, H. (1973). Amplification of ribosomal DNA in Aclieta. II. The number of nucleotide pairs of the chromosomes and chromomeres involved in amplification. Hereditas 73, LIMA-DE-FARIA, A., JAWORSKA, H. & DASKALOFF, S. (1973). Amplification of ribosomal DNA in Acheta. III. The release of DNA copies from chromomeres. Hereditas 73, MANDL, A. M. (1963). Preovulatory changes in the oocyte of the adult rat. Proc. R. Soc. B 158, MAULEON, P. (1967). Cinetique de l'ovog^nese chez les mammiferes. (In Proceedings of the Colloquium on Physiology and Reproduction in Mammals, Paris, 1966.) Archs Anat. microsc. Morph. exp. 56, Suppl. 3-4, MERCHANT, H. & CHANC, H. C. (1971). An electron microscopic study of mouse eggs matured in vivo and in vitro. Anat. Rec. 171, MILLER, O. L., BEATTY, B. R. & HAMKALO, B. A. (1972). Nuclear structure and function during amphibian oogenesis. In Oogenesis (ed. J. D. Biggers & A. E. Schuetz), pp Baltimore: University Park Press.

12 600 L. A. Chouinard MOORE, G. P. M., LLNTERN-MOORE, S., PETERS, H. & FABER, M. (1974). RNA synthesis in the mouse oocyte. J. Cell Biol. 60, OAKBERG, E. F. (1967). 3 H-uridine labelling of mouse oocytes. (In Proceedings of the Colloquium on Physiology and Reproduction in Mammals, Paris, 1966.) Arclis Anat. microsc. Morph. e.vp. 56, Suppl. 3-4, OAKBERG, E. H. (1968). Relationship between stage of follicular development and RNA synthesis in the mouse oocyte. Mutation Res. 6, PELC, S. R. (1972). Metabolic DNA in ciliated protozoa, salivary gland chromosomes, and mammalian cells. Int. Rev. Cytol. 32, PETERS, H. (1969). The development of the mouse ovary from birth to maturity. Ada endocr. 62, SORENSEN, S. A. (1973). Cinematography of mouse oocyte maturation utilizing Nomarski differential-interference microscopy. Am. J. Anat. 136, SZOLLOSI, D., CALARCO, P. & DONAHUE, R. P. (1972). Absence of centrioles in the first and second meiotic spindles of mouse oocytes. J. Cell Sci. 11, TSUDA, H. (1965). An electron microscope study of the oogenesis in the mouse, with special reference to the behavior of oogonia and oocytes at meiotic prophase. Arclwm liistol. jap. 25, WiscHNlTZER, S. (1966). The maturation of the ovum and growth of the follicle in the mouse ovary. A phase contrast microscope study. Growth 30, ZAMBONI, L. (1972), Comparative studies of the ultrastructure of mammalian oocytes. In Oogenesis (ed. J. D. Biggers & A. E. Schuetz), pp Baltimore: University Park Press. {Received 2 September 1974) Figs Photomicrographs of the mouse oocyte nucleus during the preparatory stage of antral follicle development (postnatal days 14-20); x Fig. 1, postnatal day 14; Figs. 2, 3, postnatal day 15; Figs. 4-6, postnatal day 20. Several structural components are readily identified within the nucleus during that stage: a, the nucleolus; b, the extranucleolar bodies; c, the heterochromatic knobs; cc, the condensing chromosomes.

13 Oocyte nucleus in developing antral follicle

14 602 L. A. Chouinard Figs Photomicrographs of sections of a mouse oocyte nucleus at the end of the maturative stage of antral follicle development (postnatal day 26); x The structural components of the nucleus at that time are represented by the nucleolus (a), the extranucleolar bodies (6), the heterochromatic knobs (c) and the condensing lampbrush-like chromosomes (cc). In places, the more condensed portions of the bivalents appear as more densely stained clumps of chromatin material (arrowheads).

15 Oocyte nucleus in developing antral follicle 603 ir" -C.-Y "

16 604 L. A. Chouinard Figs Photomicrographs of sections of a mouse oocyte nucleus toward the end of the preovulatory stage of antral follicle development (postnatal day 28); x The nuclear cavity is seen to contain the following structural entities: a, the nucleolus; b, the extranucleolar bodies; c, the heterochromatic knobs and cc, the condensing bivalents. The nucleolar mass is surrounded by an irregularly shaped halo of moderately condensed chromatin material. Some of the bivalents have already reached an advanced stage of condensation and are seen to be partially in contact with the undulating nuclear envelope (Figs. 13, 16, 17).

17 Oocyte nucleus in developing antral follicle 60; ) 39 CEL 17

18 606 L. A. Chouinard Figs Electron micrographs depicting some of the ultrastructural features of the formed components of the mouse oocyte nucleus during the preparatory stage of antral follicle development; x Figs. 19, 20, postnatal day 14; Fig. 21, postnatal day 16; Figs , postnatal day 20. Fig. 19. The nucleolus (a) appears as an electron-dense compact mass exhibiting no internal structure. The fine structure of the nucleolar material, because of its compactness, is not readily analysed in ordinary pale-gold sections examined at this low magnification. Grey-silver sections, however, provide enough transparency to resolve the texture of this apparently homogeneous material into a feltwork of randomly oriented fibrils 6-10 nm in diameter (inset). Arrowheads point to electronopaque granules associated with small aggregates of chromatin fibrils. Fig. 20. The extranucleolar body (b) exhibits a fibrillogranular texture and is seen to consist of intermingled masses made up of rather closely arranged convoluted fibrils, 6 10 nm in width, in which are uniformly interspersed moderately electrondense granules approximately 15 nm in diameter. Figs. 21, 22. Portions of the oocyte nucleoplasm at postnatal days 16 and 20. Electron-opaque granules (arrowheads), nm in diameter are seen, in places, to lie in close association with poorly defined aggregates of chromatin fibrils. Figs The heterochromatic knobs (c) are made up of an entanglement of closely arranged bundles of electron-dense fibrils, 8-10 nm in diameter, embedded in all ill-defined matrix of only slightly lower opacity and not readily characterized in the micrographs. The heterochromatic knob (c) seen in Fig. 25 lies over the surface of an extranucleolar body (b).

19 Oocyte nucleus in developing antral follicle Li? i^^n^m

20 608 L. A. Chouinard Figs Electron micrographs depicting some of the ultrastructural features of the formed components of the mouse oocyte nucleus at the end of the maturative follicle stage of antral follicle development (postnatal day 26); x Fig. 26. The ultrastructural features of the nucleolus (a) are indistinguishable from those observed for that same organelle at the onset of antral follicle development (cf. legend to Fig. 19). A few scattered electron-opaque grains (arrowheads) are seen within the loosely dispersed chromatin material surrounding the nucleolar mass. Figs The ultrastructural features of both the extranucleolar bodies (b) and the heterochromatic knob (c) are indistinguishable from those already described for these same organelles during the preparatory follicle stage of antral follicle development (cf. legends to Figs. 20, 23-25). In Figs. 28 and 29, the heterochromntic knob lies in close apposition to the surface of an extranucleolar body.

21 Oocyte nucleus in developing antral follicle f t ^

22 6io L. A. Chouinard Figs The less-condensed segments of the bivalents (/c) appear in the form of irregularly shaped areas made up of loosely arranged entanglements of moderately electron-dense chromatin fibrils; isolated or small aggregates of electron-opaque granules (arrowheads) are also seen here and there within these areas of lesscondensed chromatin. The more condensed portions of the bivalents (cc), on the other hand, are represented by irregularly shaped masses made up of a number of small clumps of condensing chromatin intermingled with clusters of electronopaque granules (arrowheads).

23 Oocyte nucleus in developing antral follicle 611 /c /C mf- /r 30 *!

24 612 L. A. Chouinard Figs Electron micrographs depicting some of the ultrastructural features of the formed components of the mouse oocyte nucleus toward the end of the preovulatory stage of antral follicle development (postnatal day 28); x Figs. 34, 35. The condensed portions of the bivalents (cc) appear as agglomerations of closely packed moderately electron-dense chromatin fibrils; the peripheral regions of these agglomerations are seen to consist of more loosely arranged chromatin fibrils in which are embedded a few widely scattered isolated or small clusters of electronopaque granules (arrowheads). Figs. 36, 37. A dense rounded heterochromatic knob (c) is seen to be associated with the surface of an extranucleolar body (b) in Fig. 36. The same heterochromatic knob (c) appears also to be associated, on the opposite side, by means of a narrow chromatin stalk, to the condensed portion of a bivalent (cc) in Fig. 37. Except for 2 light-staining areas within its mass, the heterochromatic knob consists of a tight dense mass of intertwined bundles of fibrils.

25 Oocyte nucleus in developing antral follicle :

26 614 L. A. Chouinard Fig. 38, and inset. The oocyte nucleolus (a) exhibits ultrastructural features similar to those already described for that nuclear organelle during the preparatory and maturative stage of antral follicle development (cf. legend to Fig. 19). The irregularly shaped halo surrounding the nucleolar body is seen to be made up of a complex meshwork of chromatin fibrils varying from a loose arrangement in some places to a dense, more closely packed one, in others; electron-opaque granules occurring singly or in clusters are also observed here and there within this chromatin halo. Fig. 39. A rounded heterochromatic knob (c), apparently unattached to the condensed portion of a bivalent is seen in close apposition to the surface of a fibrillogranular body (b). Fig. 40. A rounded heterochromatic knob (c) which lies apparently free in the nucleoplasm of the oocyte. Figs. 41, 42. Two fibrillogranular extranucleolar bodies (6) supposedly in the process of undergoing dissolution in the oocyte nucleoplasm. The compact electrondense fibrillar masses (arrowheads), of varying size and shape, seen over the surface of the shrinking extranucleolar bodies, are thought to originate from the fragmentation of a heterochromatic knob.

27 Oocyte nucleus in developing antral follicle 40 V

28