JOURNAL OF ULTRASTRUCTURE RESEARCH
61,
10- 20 (1977)
NucLear Cytochemistry of Mouse Oogenesis 1. Changes in Extranucleolar Ribonucleoprotein Components through Meiotic Prophase F. PALOMBI 1 AND A. VIRON Istituto di Istologia ed Embriologia Generale dell'Universitd di Roma, Rome, Italy, and Institut de Recherches Scientifiques sur le Cancer, Villejuif, France Received February 25, 1977 The presence of RNP-containing nuclear structures as revealed by ultrastructural cytochemistry h a s been sequentially followed during mouse oogenesis in u l t r a t h i n cryosections. Stage-specific modifications of chromatin, nucleolus, and extranucleolar components are described from the early stages of meiosis to the late p r e m a t u r a t i o n stages. In particular, changes in the population of perichromatin granules are described and confronted with the current information on RNP production during m a m m a l i a n oogenesis. The cytochemical approach has proved particularly helpful in demonstrating a unique richness in RNP-carrying particles in the nucleus of the growing oocyte, and in the identification of new RNP-carrying components at late stages of oogenesis.
The combined use of ultrastructural cytochemistry and autoradiography has in recent years provided a relevant body of information on the nature and dynamics of some ribonucleoprotein-(RNP) containing components of the interphase nucleus [see (7) for a review]. Among these, perichromatin fibrils, particularly evident at the periphery of the condensed chromatin after EDTA bleaching (5) are perhaps the best characterized so far. They have been shown to be the rapidly labeled component after [~H]uridine incubation (11), to increase in conditions known to enhance transcription (20, 24), and to decrease after a-amanitin treatment (25), and hence are thought to contain the newly transcribed DNA-like RNA (HnRNA). More intriguing is the question of which morphological nuclear component represents the structural substrate of subsequent maturation steps of the heterogeneous nuclear RNA. A certain amount of evidence is available indicating that perichromatin granules are involved in such a process. These RNP-containing particles
(17) have been observed in structural continuity with perichromatin fibrils, and their number appears increased after treatment with drugs known to inhibit rRNA synthesis (18). Unfortunately, their small size and scattered distribution do not allow the study of their labeling kinetics, as possible for Balbiani ring granules of polytene chromosomes. Nevertheless, the close structural analogy between the two nuclear components (29) can be taken as additional indication supporting the hypothesis (17) that perichromatin granules are the morphological equivalent of the maturative forms of rapidly labeled extranucleolar nuclear RNA. On this basis, we thought that mammalian oogenesis could be an interesting model to be approached by nuclear cytochemistry. In this system, in fact, the oocyte chromosomes undergo a slow and well known sequence of configurational changes: They condense, synapse, and cross over during early, fetal stages, then decondense and assume a lampbrush-type configuration (3) during the dictyate stage lasting from birth to just prior to ovulation.
1 Part of this work has been performed during the tenure of a N.A.T.O. fellowship to F.P. at Villejuif. 10 Copyright © 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 0022-5320
NUCLEAR CYTOCHEMISTRY OF MOUSE OOGENESIS
This sequence of events and the unique condition of a continuously growing cell represent a natural model in which changes in chromosome configuration and genetic activity can be related to the pattern of nuclear RNP components. MATERIALS AND METHODS For a complete stage sequence, ovaries from fetal, juvenile, and adult Swiss mice were used. Oocytes at leptotene to pachytene meiotic stages were studied from fetuses of 16 and 18 days and in newborn anireals. Ovaries of young specimens (8-, 14-, and 21day-old mice) were used for small and medium oocytes, contained, respectively, in monolaminar and p l u r i l a m i n a r follicles as well as in follicles entering a n t r a l stages; adult (2- to 3-month-old) cycling anireals were the source of the large oocytes contained in a n t r a l follicles. After m a n y attempts with Epon-embedded material, it proved best to work on cryosections, applying a modification (26) of Bernhard's EDTA regressive method (5). Conventional dehydration, embedding, and contrasting procedures were used on the contralateral fetal ovaries, to check the precise meiotic stage at each day of development. The dissected ovaries were immersed in cold 2.5% glutaraldehyde in 0.1 M cacodylate buffer; large oocytes from a n t r a l follicles were also fixed in the same fixative immediately after mechanical isolation in phosphate-buffered saline (PBS). After a l-hr fixation and overnight rinsing in buffer, the specimens were encapsulated in 20% gelatin or in bovine serum albumin (BSA) (•4); after a 15-rain immersion in glycerol to prevent ice crystal formation, freezing was performed by quick immersion in liquid nitrogen. Thin frozen sections were cut with a Sorvall Cryokit adapted to a Porter-Blum MT2 ultramicrotome, collected on plastic rings from a surface of DMSO, rapidly rinsed in a drop of distilled water, and t h e n passed onto a Formvar carboncoated copper grid. The grid was subsequently passed for 10 sec on a drop of 5% aqueous uranyl acetate, t h e n for 2 rain in 2% H202, t h e n kept for 4 rain in the dark on 2.5% uranyl acetate in sodium citrate (pH 5.5) (26), and finally contrasted with Reynold's lead citrate. Observation was performed on Siemens 1A and 101 electron microscopes. RESULTS
Bleaching of condensed chromatin by either EDTA or citrate in uranyl contrasted nuclei allows identification of a number of extranucleolar RNP-containing structures. In our material, because of the high degree of chromatin despiralization
11
in most stages, best contrast was obtained by regressive staining of frozen thin sections. This procedure has on the one hand the limitation of giving poor resolution of fine details such as perichromatin fibrils and nucleolar granules, but on the other hand yields better tissue preservation allowing the detection of a much richer population of perichromatin granules of uneven size (26). As expected, predictyate stages of oogenesis were found to proceed synchronously in the fetal ovaries, a condition which facilitates stage identification at a time when follicles have not yet formed. Leptotene oocytes were found to represent most of the germ cell population at day 16 of fetal development, as evidenced, in conventionally stained Epon sections of the contralateral ovary, by the presence of chromosomal cores (12). This cell type, characterized by a high nucleoplasmatic ratio, displays widely distributed bleached chromatin (Fig. 1) and an appreciable amount of perichromatin granules. The nucleolus at this stage is represented by multiple small bodies of very simple structure (Fig. 1). Pachytene stage, as identified by the presence in control sections of synaptinemal complexes (12), is a long-lasting stage which was found both at day 18 of fetal development and on the day of birth. During this stage cell volume increases, and a normal fibrillogranular nucleolus develops. Chromatin is apparent as wide bleached masses among which perichromatin granules are present, while a continuous net of contrasted material [interchromatin net (26)] is the most prominent RNP component (Fig. 2). Shortly after birth the synaptic stages of meiosis have been accomplished, and the oocyte enters a stage of meiotic arrest (the dictyate stage) which will last Until just prior to ovulation or atresia. At the same time, the first wave of follicles is observed to enter follicular growth. From this time on, while the majority of oocytes are represented by nongrowing cells (resting oo-
FIG. 1. Oocyte at leptotene stage. In the nucleus, wide areas of condensed chromatin (ch) appear bleached; among these, perichromatin granules (pg) represent the main extranucleolar contrasted component. Two poorly developed nucleolar structures (n) of very simple composition are observable. × 21 000. FIG. 2. Oocyte at pachytene stage. The characteristic development of a continuous net of contrasted material (interchromatin net, in) is observed. × 18 000. 12
NUCLEAR CYTOCHEMISTRY OF MOUSE OOGENESIS
cytes) contained in flat monolaminar follicles (primordial follicles), a certain number of them appear engaged in cell growth, paralleled by follicular development (23). In the nucleus of the ~resting" oocyte (Fig. 3) the interchromatin net characteristic of the pachytene stage is greatly reduced, and the areas of bleached chromatin appear less defined. On the other hand, perichromatin granule concentration and nucleolar structure appear unmodified. In those oocytes that have entered the growth phase and belong to follicles engaged in follicular growth a significantly modified pattern is observed, consisting mainly of a striking enrichment in perichromatin granules; the visual evidence of increased concentration of perichromatin granules at this stage (Figs. 4-6) is indicative of a n even higher increase in absolute number since nuclear volume increases during follicular development. A marked reduction of the bleached areas is well apparent at these stages, indicating that they are poor in condensed chromatin. Nucleolar structure appears also highly modified at these follicular stages. Vacuoles are in fact observed within the large round nucleolus in the process of enriching in fibrillar islands (9) which appear well contrasted (Figs. 5 and 6). At a time when the largest plurilaminar follicles begin to develop an antral cavity, oocyte nuclear components appear again highly modified; while a certain amount of bleached condensed chromatin starts to reappear, the enormous amount of perichromatin granules appears greatly reduced (Fig. 7), while a new RNP-containing component has, on the other hand, formed as a strongly contrasted round structure of compact texture (dense extranucleolar structures, Fig. 7). At this stage the nucleolus has developed into a homogeneous dense sphere, an aspect which will persist (Fig. 8) until dissolution takes place at the time of meiosis resumption. The completely developed antral follicle,
13
which contains a fully grown oocyte about to enter maturation, was studied in mature adult animals, since this stage is absent (or found only in the process of atresia) in the prepubertal animal. In the oocyte nucleus clumps of bleached chromatin are now well apparent both at the periphery of the prominent nucleolus and in contact with the nuclear envelope (Fig. 8). As in the previous stage (Fig. 7), dense extranucleolar structures are again found (Fig. 9), while a remarkable and intriguing change has occurred with regard to perichromatin granules; the heterogeneous population of granules observed so far at any stage, and found to reduce at the early antral stage, has now disappeared as such, to be replaced, throughout the nucleoplasm, by a multiplicity of extremely fine grains, the low contrast of which gives the nucleoplasm a general feature of fine granularity (Fig. 8). Moreover, at the border of the bleached clumps of condensed chromatin, a new population of granules has appeared, in the form of very contrasted coarse grains, arranged in clusters (Fig. 10). DISCUSSION
Through the long meiotic prophase, the primary oocyte undergoes a series of well known changes in chromatin configuration and nucleolar structure, as well as a striking increase in size. All of these changes are characteristic of definite stages, the sequence of which can be reconstructed by means of morphological parameters (for predictyate stages) and on the basis of cell size and growth of the follicle (for dictyate stages). Our cytochemical approach, which has the advantage of viewing the regressively contrasted RNP structures in the nucleus at the same time as the bleached chromatin (26), has revealed that the concentration and classes of RNP particles in the oocyte nucleus undergo apparent changes which are stage specific (Fig. 11). We intend to critically confront the structural
Fias. 3 AND 4. Changes in oocyte nucleoplasm before and after the beginning of follicular growth. FIG. 3. Nucleoplasm of"resting" oocyte: bleached chromatin (ch) and perichromatin granules (pg) are scattered throughout the nucleus, n, Nucleolus. × 17 000. FIG. 4. Nucleus of oocyte in bilaminar follicle: The nucleoplasm is enriched in perichromatin granules, while the areas occupied by clumps of bleached chromatin are reduced, n, Nucleolus. x 17 000. 14
FIG. 5. Growing oocyte in a trilaminar follicle. The nucleoplasm is populated by a strikingly high amount of perichromatin granules of heterogeneous size. Restricted areas are occupied by clumps of condensed chromatin (ch). In the very prominent nucleolus (n), vacuoles are enriching in dense fibrillar islands (fi). x 12 000. 15
16
PALOMBI AND VIRON
FIG. 6. Nucleus of oocyte in p l u r i l a m i n a r follicle. The nucleoplasm appears highly enriched in perichrom a t i n granules, and poor in condensed chromatin. The nucleolus (n) is transforming into an homogeneously dense structure, x 17 000.
dynamics observed with the current information on genetic activity at the single stages, in order to verify to what extent the observed changes can legitimately be read in functional terms. In the nuclei studied perichromatin granules are the extranucleolar RNP component that can provide more indications, since the nature of the other described extranucleolar RNP structures is undefined so far. A certain amount of evidence from morphological (29) and cytochemical (17) studies as well as from the use of inhibitors such as lasiocarpine, aflatoxin (18), and cordycepin (27) seem in fact to indicate, though not definitively [for a review, see (7)], that perichromatin granules are the morphological substrate of some form of pre-messenger RNA. In this respect the primary oocyte has proved to be the most suitable of models for cytochemical studies, since such apparent changes in perichromatin
granule population (Fig. 11) have not been observed to occur either naturally or after experimental treatment in any other system. At the early meiotic stages, relevant amounts of bleached chromatin characterize leptotene and pachytene stages, corresponding to the formation of meiotic bivalents; the presence, among the bleached condensing chromosomes, of RNP-containing structures from the very onset of meiotic prophase (leptotene, Fig. 1) suggests some level of gene expression at this early stage. In the literature, [3H]uridine incorporation has been observed at leptotene (4) as well as at pachytene, (2, 4) stages ofmammalian oogenesis, and labeling was reported to decrease from leptotene to pachytene (4). In our material, the similar concentration of perichromatin granules observed at the two stages as well as the
NUCLEAR CYTOCHEMISTRY OF MOUSE OOGENESIS
17
Fro. 7. Nucleus of oocyte at early a n t r a l stage. With a n t r u m tbrmation, perichromatin granule population appears impoverished while chromatin (ch) starts to recondense; dense extranucleolar structures (es) are characteristically present at antra-1 stages. × 24 000.
characteristic formation, at pachytene, of an interchromatin net known to Contain ribonucleoproteins (26) suggests that cell growth occurring from leptotene to pachytene is accompanied, as in the male line (13, 16), by a rich production of RNP. The slow evolution of a fully developed nucleolus from the multiple small nucleolar structures of leptotene is analogous to the slow process of nucleologenesis observed in rat oocytes (22). The postdiplotene oocyte which remains in a stage of meiotic arrest until just prior to ovulation is characterized by a particular chromosome configuration, the "dictyate" condition (3) analogous to the lampbrush state found in amphibia. As for genetic activity of the dictyate oocyte during the resting and growing periods, it was observed (1, 2, 19, 21) that increasingly high levels of RNA precursors are incorporated from resting to plurilami-
nar stages of folliculogenesis both in the nucleolus and in the nucleoplasm, maximum values of activity corresponding to large oocytes contained in plurilaminar follicles. Our observations indicate that during the resting and growth phases two main features are apparent: the increasing degree of chromatin despiralization and the change from low to strikingly high concentrations of perichromatin granules (Figs. 4-6). Both findings are in accordance with the expectations from the lampbrush configuration of chromosomes and the increasing levels of incorporation of RNA precursors, thus lending further support to the view that perichromatin granules are the expression of transcription. It is moreover, conceivable that the observed enrichmerit in perichromatin granules also reflects an accumulation of transcription product, resulting from delayed dismissal
FIGS. 8-10. Nucleus of oocyte at late antral stage. FIG. 8. Condensed chromatin (ch) is apparent around the nucleolus (n) and the nuclear membrane. At the border of condensed chromatin regions, clusters of coarse grains (cg) are apparent. The nucleoplasm is populated by a multiplicity of very fine granules, while the typically heterogeneous population of perichromatin granules is missing. × 16 000. FIo. 9. Dense extranucleolar structure. × 30 000. 18
19
NUCLEAR CYTOCHEMISTRY OF MOUSE OOGENESIS
to the cytoplasm, as already suggested for the amphibian oocyte (8). As for the nucleolus of the growing oocyte, the observation that the intranucleolar islands and the adjacent parts of the nucleolus are not bleached, but well contrasted (Fig. 5), seems to disprove the previous hypothesis (9) that these represent regions of ribosomal gene amplification. While there is general agreement on the pattern of genetic activity in oocytes at preantral stages of follicular growth, much more discussed and intriguing is the issue of transcription at later, i.e., antral, stages, in which oocyte growth has arrested (19). Recent autoradiographic experiments (28) have demonstrated that, contrary to previous concepts (19, 21), RNA is synthesized throughout the antral stages of meiotic prophase. As for the levels of this synthesis, the only reliable direct datum comes from autoradiographic experiments after injection of labeled precursor into the antral cavity of the largest type of antral follicles (30), demonstrating that the latest period preceding maturation is very active in transcription. On the other hand, the levels of RNA polymerase activity, as cytochemically detected, have been reported to decrease with the progression of antral stages (19), and oocytes well beyond the growth period have been shown to contain chromosomes no longer in a typical lampbrush configuration (15). In the absence of direct quantitative data on the complete stage sequence, the whole of these observations could perhaps be taken as indicating that transcription slows down at antral stages, but that, at the latest steps, in preparation for maturation, a new rise in transcriptional activity takes place. In accordance with this hypothesis, in oocytes isolated in vitro, maturation (i.e., resumption of meiosis) has been observed (6) to be preceded by and to be dependent upon a late wave of transcription. Fm. 10. Clusters of coarse grains. × 30 000.
nucleus of oocyte at corresponding stages
B
stages of follicular development
o
G E
FIG. 11. Diagrammatic representation of the changes in perichromatin granule population observed in the oocyte nucleus during follicular growth. Apparent changes in concentration occur at the onset of follicular growth and at a n t r u m formation. The qualitative change observed in the oocytes approaching m a t u r a t i o n (see text) is also shown. A, U n i l a m i n a r resting follicle. B, C, D, Bilaminar, trilaminar, and plurilaminar stages of p r e a n t r a l development. E, Early a n t r a l stage. F, Late a n t r a l stage.
PALOMBI AND VIRON
20
L. L., J. Cell Sci. 4, 655 (1969). Our cytochemical observations seem to 3. BAKER,T. G., AND FRANCHI,L. L., Chromosoma support the above discussed hypothesis on 22, 358 (1967). the pattern of gene activity during antral 4. BAKKEN,A. H., J. Cell Biol., 70, 144a (1976). stages. First of all, in fact, a marked de5. BERNHARD, W., J. Ultrastruct. Res. 27, 270 crease in concentration of perichromatin (]969). 6. BLOOM,A. M., AND MUKHERJEE,B. B., Exp. Cell granules is observed at early antral stages Res. 74, 577 (1972). (Fig. 7). Secondly, at late antral stages the 7. BOUTEILLE,M., LAVAL,M., ANDDUPUY-CoIN,A. nucleus appears repopulated by a relevant M. in BUSCH, H. (Ed.), The Cell Nucleus, Vol. amount of fine granules (Figs. 8-10) which I, p. 3. Academic Press, N e w York and London, 1974. we regard as corresponding to the ~finest 8. CALLAN, H. G. in LIMA-DE-FARIA, A. (Ed.), elements of the usual heterogeneous popuHandbook of Molecular Cytology, p. 540. lation; assuming that these represent North-Holland, Amsterdam and London, newly synthesized, immature, perichro1969. matin granules (26), we would have visu9. CHOUINARD,L. A., J. Cell Sci. 9, 637 (1971). alized the slowing down of oocyte genetic 10. CHOUINARD,L. A., J. Cell Sci. ]7, 589 (1975). activity at early antral stages, and the late 11. FAKAN, S., AND BERNHARD,W., Exp. Cell Res. 67, 129 (1971). switching on of a new transcription wave, 12. FRANCHI, L. L., AND MANDL, A. M., Proc. R. in preparation for maturation. Soc. (Lond.) Ser. B. 157, 99 (1962). Other qualitative changes observed for 13. KIERSZENBAUM,A. L., AND TRES, L. L. J. Cell Biol. 60, 39 (1974). RNP nuclear components in antral oocytes 14. KUHLMANN, W. D., AND VIRON, A., J. Ultraare the presence of extranucleolar dense struct. Res. 41, 385 (1972). structures (Fig. 7 and 9) through antral 15. MILLER, O. L., AND BAKKEN,A. H., in Karolinstages, and the appearing, during late anska Symposium on Research Methods in Retral development, of clusters of coarse production and Endocrinology, Vol. 5, p. 155 (1968). grains (Figs. 8 and 10) in association with condensing chromatin. Both new struc- 16. MONESI, V., J. Cell Biol. 22, 521 (1964). tures had already been observed in con- 17. MONNERON,A,, AND BERNHARD, W., J. Ultrastruct. Res. 27, 266 (1969). ventional preparations (10), but their na18. MONNERON,A., LAFARGE,C., AND FRAYSSINET, ture of RNA carriers was not known. C., C. R. Acad. Sci. Paris 267, 2053 (1968). Though the functional role of these stage- 19. MOORE, G. P. M., LINTERN-MOORE,S., PETERS, specific changes remains an open problem, H., AND FABER, M.,J. CellBiol. 60, 416 (1974). we think that the overall changes ob- 20. NASH, R. E., PUVION,E., ANDBERNHARD,W., J. Ultrastruct. Res. 53, 395 (1975). served at antrum formation and at late 21. OAKEERG, E. F., Mutat. Res. 6, 155 (1968). prematuration stages can be considered as 22. PALOMEI,F., ANDSTEFANINI,M., J. Ultrastruct. indicative of some new event concerning Res. 47, 61 (1974). RNA production, storage, or transport. 23. PETERS, H., Aeta Endocrinol. 62, 98 (1969). This work was performed in part under CNR Research Project Biology of Reproduction (grant No. 760.0300.85) and was supported in part by grant No. 730.0208 from the Ford Foundation. We wish to express our gratitude to Prof. W. Bernhard for constant help and encouragement during the course of this study, and to thank Prof. M. Stefanini and Dr, F. Mangia for advice and discussion during the preparation of the manuscript. We are also indebted to Miss I. Weigert for the preparation of Fig. 11. REFERENCES 1. BACHVAROVA,R., Develop. Biol. 40, 52 (1974). 2. BAKER, T. G., BEAUMONT, H. M., AND FRANCHI,
24. PETROV,P., AND BERNHARD,W., J. Ultrastruct. Res. 35, 386 (1971). 25. PETROV,P., AND SEKERIS, C. E., Exp. Cell Res. 69, 393 (1971). 26. PUVION,E., ANDBERNHARD,W., J. Cell Biol. 67, 200 (1975). 27. PUVION,E., MOYNE, G., AND BERNHARD,W., J. Microsc. Biol. Cell. 25, 17 (1976). 28. RODMAN, T. C., AND BACHVAROVAR., J. Cell Biol. 70, 251 (1976). 29. VAZQUEZ-NIN,G., ANDBERNHARD,W., J. Ultrastruct. Res. 36, 842 (1971). 30. WASSARMAN,P. M., AND LETOURNEAU,G. E., Nature (Lond.) 261, 73 (1976).
61,
10- 20 (1977)
NucLear Cytochemistry of Mouse Oogenesis 1. Changes in Extranucleolar Ribonucleoprotein Components through Meiotic Prophase F. PALOMBI 1 AND A. VIRON Istituto di Istologia ed Embriologia Generale dell'Universitd di Roma, Rome, Italy, and Institut de Recherches Scientifiques sur le Cancer, Villejuif, France Received February 25, 1977 The presence of RNP-containing nuclear structures as revealed by ultrastructural cytochemistry h a s been sequentially followed during mouse oogenesis in u l t r a t h i n cryosections. Stage-specific modifications of chromatin, nucleolus, and extranucleolar components are described from the early stages of meiosis to the late p r e m a t u r a t i o n stages. In particular, changes in the population of perichromatin granules are described and confronted with the current information on RNP production during m a m m a l i a n oogenesis. The cytochemical approach has proved particularly helpful in demonstrating a unique richness in RNP-carrying particles in the nucleus of the growing oocyte, and in the identification of new RNP-carrying components at late stages of oogenesis.
The combined use of ultrastructural cytochemistry and autoradiography has in recent years provided a relevant body of information on the nature and dynamics of some ribonucleoprotein-(RNP) containing components of the interphase nucleus [see (7) for a review]. Among these, perichromatin fibrils, particularly evident at the periphery of the condensed chromatin after EDTA bleaching (5) are perhaps the best characterized so far. They have been shown to be the rapidly labeled component after [~H]uridine incubation (11), to increase in conditions known to enhance transcription (20, 24), and to decrease after a-amanitin treatment (25), and hence are thought to contain the newly transcribed DNA-like RNA (HnRNA). More intriguing is the question of which morphological nuclear component represents the structural substrate of subsequent maturation steps of the heterogeneous nuclear RNA. A certain amount of evidence is available indicating that perichromatin granules are involved in such a process. These RNP-containing particles
(17) have been observed in structural continuity with perichromatin fibrils, and their number appears increased after treatment with drugs known to inhibit rRNA synthesis (18). Unfortunately, their small size and scattered distribution do not allow the study of their labeling kinetics, as possible for Balbiani ring granules of polytene chromosomes. Nevertheless, the close structural analogy between the two nuclear components (29) can be taken as additional indication supporting the hypothesis (17) that perichromatin granules are the morphological equivalent of the maturative forms of rapidly labeled extranucleolar nuclear RNA. On this basis, we thought that mammalian oogenesis could be an interesting model to be approached by nuclear cytochemistry. In this system, in fact, the oocyte chromosomes undergo a slow and well known sequence of configurational changes: They condense, synapse, and cross over during early, fetal stages, then decondense and assume a lampbrush-type configuration (3) during the dictyate stage lasting from birth to just prior to ovulation.
1 Part of this work has been performed during the tenure of a N.A.T.O. fellowship to F.P. at Villejuif. 10 Copyright © 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 0022-5320
NUCLEAR CYTOCHEMISTRY OF MOUSE OOGENESIS
This sequence of events and the unique condition of a continuously growing cell represent a natural model in which changes in chromosome configuration and genetic activity can be related to the pattern of nuclear RNP components. MATERIALS AND METHODS For a complete stage sequence, ovaries from fetal, juvenile, and adult Swiss mice were used. Oocytes at leptotene to pachytene meiotic stages were studied from fetuses of 16 and 18 days and in newborn anireals. Ovaries of young specimens (8-, 14-, and 21day-old mice) were used for small and medium oocytes, contained, respectively, in monolaminar and p l u r i l a m i n a r follicles as well as in follicles entering a n t r a l stages; adult (2- to 3-month-old) cycling anireals were the source of the large oocytes contained in a n t r a l follicles. After m a n y attempts with Epon-embedded material, it proved best to work on cryosections, applying a modification (26) of Bernhard's EDTA regressive method (5). Conventional dehydration, embedding, and contrasting procedures were used on the contralateral fetal ovaries, to check the precise meiotic stage at each day of development. The dissected ovaries were immersed in cold 2.5% glutaraldehyde in 0.1 M cacodylate buffer; large oocytes from a n t r a l follicles were also fixed in the same fixative immediately after mechanical isolation in phosphate-buffered saline (PBS). After a l-hr fixation and overnight rinsing in buffer, the specimens were encapsulated in 20% gelatin or in bovine serum albumin (BSA) (•4); after a 15-rain immersion in glycerol to prevent ice crystal formation, freezing was performed by quick immersion in liquid nitrogen. Thin frozen sections were cut with a Sorvall Cryokit adapted to a Porter-Blum MT2 ultramicrotome, collected on plastic rings from a surface of DMSO, rapidly rinsed in a drop of distilled water, and t h e n passed onto a Formvar carboncoated copper grid. The grid was subsequently passed for 10 sec on a drop of 5% aqueous uranyl acetate, t h e n for 2 rain in 2% H202, t h e n kept for 4 rain in the dark on 2.5% uranyl acetate in sodium citrate (pH 5.5) (26), and finally contrasted with Reynold's lead citrate. Observation was performed on Siemens 1A and 101 electron microscopes. RESULTS
Bleaching of condensed chromatin by either EDTA or citrate in uranyl contrasted nuclei allows identification of a number of extranucleolar RNP-containing structures. In our material, because of the high degree of chromatin despiralization
11
in most stages, best contrast was obtained by regressive staining of frozen thin sections. This procedure has on the one hand the limitation of giving poor resolution of fine details such as perichromatin fibrils and nucleolar granules, but on the other hand yields better tissue preservation allowing the detection of a much richer population of perichromatin granules of uneven size (26). As expected, predictyate stages of oogenesis were found to proceed synchronously in the fetal ovaries, a condition which facilitates stage identification at a time when follicles have not yet formed. Leptotene oocytes were found to represent most of the germ cell population at day 16 of fetal development, as evidenced, in conventionally stained Epon sections of the contralateral ovary, by the presence of chromosomal cores (12). This cell type, characterized by a high nucleoplasmatic ratio, displays widely distributed bleached chromatin (Fig. 1) and an appreciable amount of perichromatin granules. The nucleolus at this stage is represented by multiple small bodies of very simple structure (Fig. 1). Pachytene stage, as identified by the presence in control sections of synaptinemal complexes (12), is a long-lasting stage which was found both at day 18 of fetal development and on the day of birth. During this stage cell volume increases, and a normal fibrillogranular nucleolus develops. Chromatin is apparent as wide bleached masses among which perichromatin granules are present, while a continuous net of contrasted material [interchromatin net (26)] is the most prominent RNP component (Fig. 2). Shortly after birth the synaptic stages of meiosis have been accomplished, and the oocyte enters a stage of meiotic arrest (the dictyate stage) which will last Until just prior to ovulation or atresia. At the same time, the first wave of follicles is observed to enter follicular growth. From this time on, while the majority of oocytes are represented by nongrowing cells (resting oo-
FIG. 1. Oocyte at leptotene stage. In the nucleus, wide areas of condensed chromatin (ch) appear bleached; among these, perichromatin granules (pg) represent the main extranucleolar contrasted component. Two poorly developed nucleolar structures (n) of very simple composition are observable. × 21 000. FIG. 2. Oocyte at pachytene stage. The characteristic development of a continuous net of contrasted material (interchromatin net, in) is observed. × 18 000. 12
NUCLEAR CYTOCHEMISTRY OF MOUSE OOGENESIS
cytes) contained in flat monolaminar follicles (primordial follicles), a certain number of them appear engaged in cell growth, paralleled by follicular development (23). In the nucleus of the ~resting" oocyte (Fig. 3) the interchromatin net characteristic of the pachytene stage is greatly reduced, and the areas of bleached chromatin appear less defined. On the other hand, perichromatin granule concentration and nucleolar structure appear unmodified. In those oocytes that have entered the growth phase and belong to follicles engaged in follicular growth a significantly modified pattern is observed, consisting mainly of a striking enrichment in perichromatin granules; the visual evidence of increased concentration of perichromatin granules at this stage (Figs. 4-6) is indicative of a n even higher increase in absolute number since nuclear volume increases during follicular development. A marked reduction of the bleached areas is well apparent at these stages, indicating that they are poor in condensed chromatin. Nucleolar structure appears also highly modified at these follicular stages. Vacuoles are in fact observed within the large round nucleolus in the process of enriching in fibrillar islands (9) which appear well contrasted (Figs. 5 and 6). At a time when the largest plurilaminar follicles begin to develop an antral cavity, oocyte nuclear components appear again highly modified; while a certain amount of bleached condensed chromatin starts to reappear, the enormous amount of perichromatin granules appears greatly reduced (Fig. 7), while a new RNP-containing component has, on the other hand, formed as a strongly contrasted round structure of compact texture (dense extranucleolar structures, Fig. 7). At this stage the nucleolus has developed into a homogeneous dense sphere, an aspect which will persist (Fig. 8) until dissolution takes place at the time of meiosis resumption. The completely developed antral follicle,
13
which contains a fully grown oocyte about to enter maturation, was studied in mature adult animals, since this stage is absent (or found only in the process of atresia) in the prepubertal animal. In the oocyte nucleus clumps of bleached chromatin are now well apparent both at the periphery of the prominent nucleolus and in contact with the nuclear envelope (Fig. 8). As in the previous stage (Fig. 7), dense extranucleolar structures are again found (Fig. 9), while a remarkable and intriguing change has occurred with regard to perichromatin granules; the heterogeneous population of granules observed so far at any stage, and found to reduce at the early antral stage, has now disappeared as such, to be replaced, throughout the nucleoplasm, by a multiplicity of extremely fine grains, the low contrast of which gives the nucleoplasm a general feature of fine granularity (Fig. 8). Moreover, at the border of the bleached clumps of condensed chromatin, a new population of granules has appeared, in the form of very contrasted coarse grains, arranged in clusters (Fig. 10). DISCUSSION
Through the long meiotic prophase, the primary oocyte undergoes a series of well known changes in chromatin configuration and nucleolar structure, as well as a striking increase in size. All of these changes are characteristic of definite stages, the sequence of which can be reconstructed by means of morphological parameters (for predictyate stages) and on the basis of cell size and growth of the follicle (for dictyate stages). Our cytochemical approach, which has the advantage of viewing the regressively contrasted RNP structures in the nucleus at the same time as the bleached chromatin (26), has revealed that the concentration and classes of RNP particles in the oocyte nucleus undergo apparent changes which are stage specific (Fig. 11). We intend to critically confront the structural
Fias. 3 AND 4. Changes in oocyte nucleoplasm before and after the beginning of follicular growth. FIG. 3. Nucleoplasm of"resting" oocyte: bleached chromatin (ch) and perichromatin granules (pg) are scattered throughout the nucleus, n, Nucleolus. × 17 000. FIG. 4. Nucleus of oocyte in bilaminar follicle: The nucleoplasm is enriched in perichromatin granules, while the areas occupied by clumps of bleached chromatin are reduced, n, Nucleolus. x 17 000. 14
FIG. 5. Growing oocyte in a trilaminar follicle. The nucleoplasm is populated by a strikingly high amount of perichromatin granules of heterogeneous size. Restricted areas are occupied by clumps of condensed chromatin (ch). In the very prominent nucleolus (n), vacuoles are enriching in dense fibrillar islands (fi). x 12 000. 15
16
PALOMBI AND VIRON
FIG. 6. Nucleus of oocyte in p l u r i l a m i n a r follicle. The nucleoplasm appears highly enriched in perichrom a t i n granules, and poor in condensed chromatin. The nucleolus (n) is transforming into an homogeneously dense structure, x 17 000.
dynamics observed with the current information on genetic activity at the single stages, in order to verify to what extent the observed changes can legitimately be read in functional terms. In the nuclei studied perichromatin granules are the extranucleolar RNP component that can provide more indications, since the nature of the other described extranucleolar RNP structures is undefined so far. A certain amount of evidence from morphological (29) and cytochemical (17) studies as well as from the use of inhibitors such as lasiocarpine, aflatoxin (18), and cordycepin (27) seem in fact to indicate, though not definitively [for a review, see (7)], that perichromatin granules are the morphological substrate of some form of pre-messenger RNA. In this respect the primary oocyte has proved to be the most suitable of models for cytochemical studies, since such apparent changes in perichromatin
granule population (Fig. 11) have not been observed to occur either naturally or after experimental treatment in any other system. At the early meiotic stages, relevant amounts of bleached chromatin characterize leptotene and pachytene stages, corresponding to the formation of meiotic bivalents; the presence, among the bleached condensing chromosomes, of RNP-containing structures from the very onset of meiotic prophase (leptotene, Fig. 1) suggests some level of gene expression at this early stage. In the literature, [3H]uridine incorporation has been observed at leptotene (4) as well as at pachytene, (2, 4) stages ofmammalian oogenesis, and labeling was reported to decrease from leptotene to pachytene (4). In our material, the similar concentration of perichromatin granules observed at the two stages as well as the
NUCLEAR CYTOCHEMISTRY OF MOUSE OOGENESIS
17
Fro. 7. Nucleus of oocyte at early a n t r a l stage. With a n t r u m tbrmation, perichromatin granule population appears impoverished while chromatin (ch) starts to recondense; dense extranucleolar structures (es) are characteristically present at antra-1 stages. × 24 000.
characteristic formation, at pachytene, of an interchromatin net known to Contain ribonucleoproteins (26) suggests that cell growth occurring from leptotene to pachytene is accompanied, as in the male line (13, 16), by a rich production of RNP. The slow evolution of a fully developed nucleolus from the multiple small nucleolar structures of leptotene is analogous to the slow process of nucleologenesis observed in rat oocytes (22). The postdiplotene oocyte which remains in a stage of meiotic arrest until just prior to ovulation is characterized by a particular chromosome configuration, the "dictyate" condition (3) analogous to the lampbrush state found in amphibia. As for genetic activity of the dictyate oocyte during the resting and growing periods, it was observed (1, 2, 19, 21) that increasingly high levels of RNA precursors are incorporated from resting to plurilami-
nar stages of folliculogenesis both in the nucleolus and in the nucleoplasm, maximum values of activity corresponding to large oocytes contained in plurilaminar follicles. Our observations indicate that during the resting and growth phases two main features are apparent: the increasing degree of chromatin despiralization and the change from low to strikingly high concentrations of perichromatin granules (Figs. 4-6). Both findings are in accordance with the expectations from the lampbrush configuration of chromosomes and the increasing levels of incorporation of RNA precursors, thus lending further support to the view that perichromatin granules are the expression of transcription. It is moreover, conceivable that the observed enrichmerit in perichromatin granules also reflects an accumulation of transcription product, resulting from delayed dismissal
FIGS. 8-10. Nucleus of oocyte at late antral stage. FIG. 8. Condensed chromatin (ch) is apparent around the nucleolus (n) and the nuclear membrane. At the border of condensed chromatin regions, clusters of coarse grains (cg) are apparent. The nucleoplasm is populated by a multiplicity of very fine granules, while the typically heterogeneous population of perichromatin granules is missing. × 16 000. FIo. 9. Dense extranucleolar structure. × 30 000. 18
19
NUCLEAR CYTOCHEMISTRY OF MOUSE OOGENESIS
to the cytoplasm, as already suggested for the amphibian oocyte (8). As for the nucleolus of the growing oocyte, the observation that the intranucleolar islands and the adjacent parts of the nucleolus are not bleached, but well contrasted (Fig. 5), seems to disprove the previous hypothesis (9) that these represent regions of ribosomal gene amplification. While there is general agreement on the pattern of genetic activity in oocytes at preantral stages of follicular growth, much more discussed and intriguing is the issue of transcription at later, i.e., antral, stages, in which oocyte growth has arrested (19). Recent autoradiographic experiments (28) have demonstrated that, contrary to previous concepts (19, 21), RNA is synthesized throughout the antral stages of meiotic prophase. As for the levels of this synthesis, the only reliable direct datum comes from autoradiographic experiments after injection of labeled precursor into the antral cavity of the largest type of antral follicles (30), demonstrating that the latest period preceding maturation is very active in transcription. On the other hand, the levels of RNA polymerase activity, as cytochemically detected, have been reported to decrease with the progression of antral stages (19), and oocytes well beyond the growth period have been shown to contain chromosomes no longer in a typical lampbrush configuration (15). In the absence of direct quantitative data on the complete stage sequence, the whole of these observations could perhaps be taken as indicating that transcription slows down at antral stages, but that, at the latest steps, in preparation for maturation, a new rise in transcriptional activity takes place. In accordance with this hypothesis, in oocytes isolated in vitro, maturation (i.e., resumption of meiosis) has been observed (6) to be preceded by and to be dependent upon a late wave of transcription. Fm. 10. Clusters of coarse grains. × 30 000.
nucleus of oocyte at corresponding stages
B
stages of follicular development
o
G E
FIG. 11. Diagrammatic representation of the changes in perichromatin granule population observed in the oocyte nucleus during follicular growth. Apparent changes in concentration occur at the onset of follicular growth and at a n t r u m formation. The qualitative change observed in the oocytes approaching m a t u r a t i o n (see text) is also shown. A, U n i l a m i n a r resting follicle. B, C, D, Bilaminar, trilaminar, and plurilaminar stages of p r e a n t r a l development. E, Early a n t r a l stage. F, Late a n t r a l stage.
PALOMBI AND VIRON
20
L. L., J. Cell Sci. 4, 655 (1969). Our cytochemical observations seem to 3. BAKER,T. G., AND FRANCHI,L. L., Chromosoma support the above discussed hypothesis on 22, 358 (1967). the pattern of gene activity during antral 4. BAKKEN,A. H., J. Cell Biol., 70, 144a (1976). stages. First of all, in fact, a marked de5. BERNHARD, W., J. Ultrastruct. Res. 27, 270 crease in concentration of perichromatin (]969). 6. BLOOM,A. M., AND MUKHERJEE,B. B., Exp. Cell granules is observed at early antral stages Res. 74, 577 (1972). (Fig. 7). Secondly, at late antral stages the 7. BOUTEILLE,M., LAVAL,M., ANDDUPUY-CoIN,A. nucleus appears repopulated by a relevant M. in BUSCH, H. (Ed.), The Cell Nucleus, Vol. amount of fine granules (Figs. 8-10) which I, p. 3. Academic Press, N e w York and London, 1974. we regard as corresponding to the ~finest 8. CALLAN, H. G. in LIMA-DE-FARIA, A. (Ed.), elements of the usual heterogeneous popuHandbook of Molecular Cytology, p. 540. lation; assuming that these represent North-Holland, Amsterdam and London, newly synthesized, immature, perichro1969. matin granules (26), we would have visu9. CHOUINARD,L. A., J. Cell Sci. 9, 637 (1971). alized the slowing down of oocyte genetic 10. CHOUINARD,L. A., J. Cell Sci. ]7, 589 (1975). activity at early antral stages, and the late 11. FAKAN, S., AND BERNHARD,W., Exp. Cell Res. 67, 129 (1971). switching on of a new transcription wave, 12. FRANCHI, L. L., AND MANDL, A. M., Proc. R. in preparation for maturation. Soc. (Lond.) Ser. B. 157, 99 (1962). Other qualitative changes observed for 13. KIERSZENBAUM,A. L., AND TRES, L. L. J. Cell Biol. 60, 39 (1974). RNP nuclear components in antral oocytes 14. KUHLMANN, W. D., AND VIRON, A., J. Ultraare the presence of extranucleolar dense struct. Res. 41, 385 (1972). structures (Fig. 7 and 9) through antral 15. MILLER, O. L., AND BAKKEN,A. H., in Karolinstages, and the appearing, during late anska Symposium on Research Methods in Retral development, of clusters of coarse production and Endocrinology, Vol. 5, p. 155 (1968). grains (Figs. 8 and 10) in association with condensing chromatin. Both new struc- 16. MONESI, V., J. Cell Biol. 22, 521 (1964). tures had already been observed in con- 17. MONNERON,A,, AND BERNHARD, W., J. Ultrastruct. Res. 27, 266 (1969). ventional preparations (10), but their na18. MONNERON,A., LAFARGE,C., AND FRAYSSINET, ture of RNA carriers was not known. C., C. R. Acad. Sci. Paris 267, 2053 (1968). Though the functional role of these stage- 19. MOORE, G. P. M., LINTERN-MOORE,S., PETERS, specific changes remains an open problem, H., AND FABER, M.,J. CellBiol. 60, 416 (1974). we think that the overall changes ob- 20. NASH, R. E., PUVION,E., ANDBERNHARD,W., J. Ultrastruct. Res. 53, 395 (1975). served at antrum formation and at late 21. OAKEERG, E. F., Mutat. Res. 6, 155 (1968). prematuration stages can be considered as 22. PALOMEI,F., ANDSTEFANINI,M., J. Ultrastruct. indicative of some new event concerning Res. 47, 61 (1974). RNA production, storage, or transport. 23. PETERS, H., Aeta Endocrinol. 62, 98 (1969). This work was performed in part under CNR Research Project Biology of Reproduction (grant No. 760.0300.85) and was supported in part by grant No. 730.0208 from the Ford Foundation. We wish to express our gratitude to Prof. W. Bernhard for constant help and encouragement during the course of this study, and to thank Prof. M. Stefanini and Dr, F. Mangia for advice and discussion during the preparation of the manuscript. We are also indebted to Miss I. Weigert for the preparation of Fig. 11. REFERENCES 1. BACHVAROVA,R., Develop. Biol. 40, 52 (1974). 2. BAKER, T. G., BEAUMONT, H. M., AND FRANCHI,
24. PETROV,P., AND BERNHARD,W., J. Ultrastruct. Res. 35, 386 (1971). 25. PETROV,P., AND SEKERIS, C. E., Exp. Cell Res. 69, 393 (1971). 26. PUVION,E., ANDBERNHARD,W., J. Cell Biol. 67, 200 (1975). 27. PUVION,E., MOYNE, G., AND BERNHARD,W., J. Microsc. Biol. Cell. 25, 17 (1976). 28. RODMAN, T. C., AND BACHVAROVAR., J. Cell Biol. 70, 251 (1976). 29. VAZQUEZ-NIN,G., ANDBERNHARD,W., J. Ultrastruct. Res. 36, 842 (1971). 30. WASSARMAN,P. M., AND LETOURNEAU,G. E., Nature (Lond.) 261, 73 (1976).
