Correlation between germinal vesicle and oocyte development in the adult Japanese quail (Coturnix coturnix japónica). A cytochemical and autoradiographic study

According to Van Durme (1914), during the period of final rapid growth in the sparrow and swallow, the germinal vesicle always contains ‘chromatic spheres’ resembling nucleoli. Van Durme called them ‘nucléoles nucléiniens’ because he found that they later constituted the chromosomes of the first maturation spindle.

In previous work (Callebaut, 1970b) we found the existence of Feulgenpositive spherules in the central part of the germinal vesicle of the large oocytes of laying Japanese quails. In order to establish the origin and fate of these peculiar organelles, we made a cytochemical and autoradiographic study of the general evolution of the germinal vesicle during oogenesis (in the broad sense) of the adult Japanese quail.

In the present study, three groups of adult female Japanese quails were used. The first group consisted of 40 regularly laying birds receiving continuous illumination and living at an approximately constant temperature (some of them from 0 to 5°C, others from 20 to 25°C). The second group consisting of 11 adult laying Japanese quails was raised in similar conditions. These birds, however, received during their embryological life treatment with successive [³H]thymidine pulses in ovo. Through a hole in the shell over the airspace 2·2 μ Ci of [³H]thymidine 6 (10 Ci/mM) in 50 μ I distilled water were placed on the air space membrane of the egg placed in an upright position. After each application the hole in the shell was closed with adhesive cellophane tape. The first application took place in fertilized quail eggs incubated for 9·5 days at 39°C. This was followed by identical applications every 8 h. The last (seventh) application occurred in eggs incubated for 11·5 days.

The third group consisted of ten adult female quails living in temperatures varying from 0 to 5°C and in cages poorly illuminated by indirect daylight, during the short days of the Belgian winter (from November to February).

After killing the birds by decapitation the abdomen is opened and the whole set of pediculated follicles (ranging from approximately 0·5 to 19 mm) are removed from the ovary. The localization of the germinal vesicle of the oocyte is usually visible through the ovular membranes of follicles with a diameter of at least 2 mm. This site is labelled by the application of carbon particles before fixation since after the action of the fixative, the cicatricular region usually becomes invisible. After labelling, the pediculated intrafollicular oocytes and their ovaries are fixed by immersion in acetic-ethanol (1:3 v/v) for 3-4 h at room temperature, rinsed in 95% ethanol and their diameter measured with an accuracy of ± 0·5 mm (range).

They are left overnight in this solution at 4°C and the following morning, during further dehydration, the germinal disc, still covered by their thecas, is removed from the largest oocytes (3 mm diameter or more). Oocytes with a diameter of less than 3 mm are dehydrated in toto. After embedding in paraffin the oocytes and ovaries are sectioned at 7 μ m thickness.

The oocytes whose cicatricular region is already well developed and labelled with carbon particles are cut in a direction perpendicular to the plane of this region (perpendicular sections) or parallel with this plane (tangential sections). In the oocytes from which the localization of the germinal vesicle is not labelled with carbon particles (usually with a diameter of less than 2 mm) the direction of the plane of sectioning can only be approximately evaluated by the screening of serial sections. Near tangential sections of the germinal disc are found at the beginning or end of the ribbon, and near perpendicular ones in the middle part. Sections oblique to the general plane of the cicatricular region are found between these localizations. These paraffin sections are screened under the darkground microscope in order to select those containing some part of the germinal vesicle (or disintegrated germinal vesicle for mature oocytes). From oocytes with a diameter larger then 2 mm only the latter sections are employed. For enzymic digestion studies (RNase) alternating sections are placed on different slides. Enzymic digestion with RNase is performed by immersion of the deparaffinized sections in a solution of 0·2 mg RNase Sigma per ml of Tris buffer at pH 7·5 for 2 h at 37°C. With the aim of inactivating any contaminating enzyme the RNase solution is boiled for 5 min at p 4. With or without enzymic digestion the sections are stained thus: (1) with Unna’s stain; (2) with a saturated solution of fast green FCF (Matheson, Coleman & Bell) in //-butyl alcohol; (3) with the Feulgen nuclear reaction, followed sometimes by fast green FCF as in (2) above; (4) with Groat’s iron haematoxylin and eosin.

Some of the adult Japanese quails of the first or third groups receive an intraperitoneal injection of 0·7 to 1 mCi of uridine 5-³H (10 Ci/mM) or 1 to 2 mCi of [³H]thymidine 6 (14-19 Ci/mM). At intervals of 30, 60, 90 and 120 min or 3 days after this injection these birds are killed and their ovary and oocytes fixed in acetic-ethanol (l:3v/v) for 3-4h. The further histological procedure is similar to that which has been described above in the cytological study of the serial sections. These sections together with those containing similar structures from non-radioactive control oocytes (to exclude chemical background) are coated with nuclear emulsion L 4 (Ilford, England) by the dipping method. After 30-60 days exposure and photographic development the sections are stained with Groat’s iron haematoxylin and eosin.

According to the aspect of their nuclear contents the immature intraovarian oocytes of group 1 (regularly laying quails without radioactive treatment) can be classified into three stages.

The smallest oocytes found in the ovary of adult Japanese quails having a diameter from 30-50 μ m are in this stage. They contain a spherical or ovoid peripheral germinal vesicle with a diameter of 20-30 μ m. However, somewhat larger oocytes with a diameter of up to approximately 250 μ m and containing a germinal vesicle with a diameter of 40-50 μ m belong also to this stage. After staining with iron haematoxylin and eosin these oocytes are clearly seen to be in the diplotene stage of meiosis: they contain chromosomes in the form of figuresof-eight or chains due to chiasma formation, and parts of them are seen to be transversely striated. The most prominent feature in the cytoplasm of oocytes at this stage is the very voluminous paranuclear Balbiani yolk-body complex.

After the Feulgen nuclear reaction the chromosomes at this stage are seen as very fine treads on which Feulgen-positive chromatin accumulations (chromomeres) are usually visible. After staining with fast green no chromosomes can be distinguished; only very small green granules are visible homogeneously spread in the nucleoplasm. The chromosomes and the chromatin clumps localized on them are dark green after staining with Unna and the intermingled nucleoplasm is a very faint pink. In the larger oocytes (with a diameter from approximately 100 to 250 μ m), however, some chromosomes are partly or wholly red. This partial pyroninophilia probably represents a transition phase to the succeeding lampbrush chromosome stage. During this first stage, very large pyroninophilic, spherical vacuolized nucleoli, localized in the central area of the germinal vesicle among the chromosomes, can easily be seen. After RNase treatment the above-described pyroninophilic structures are no longer visible. This first developmental stage of the quail oocyte we have called the prelampbrush chromosome stage, because it precedes that of the lampbrush chromosome.

This includes oocytes with a diameter varying from 250 to approx. 1000 μ m. During the first part of this stage (consisting of oocytes with a diameter from 250 to approx. 500 μ m), the germinal vesicle is no longer found at the periphery of the oocyte but occupies a central or subcentral position in the ooplasm which now presents a homogeneous mesh-like appearance, the paranuclear Balbiani complex having disappeared. During the second part of this second stage (oocytes from approx. 500 to approx. 1000 μ m diameter) the ooplasm is no longer homogeneous and two large cytoplasmic superstructures have differentiated: (1) the central vacuolar yolk-mass; (2) the well-developed cortex which wholly surrounds this central vacuolar yolk-mass and in which a pale peripheral layer and a dense, more central layer can be discerned. After colouring with basophilic stains the latter denser layer shows an intense basophilic reaction. During this second part of the lampbrush chromosome stage the germinal vesicle again migrates or floats to the periphery of the oocyte.

A. First part of the lampbrush chromosome stage. The rapidly enlarging germinal vesicle contains lampbrush chromosomes and is stacked with innumerable very small granules. Each of these granules is usually found within a clear streak of the nucleoplasm. As found by Marza (1935) in chicken oocytes, the Feulgen reaction in the chromosomes progressively weakens as the oocyte increases in size. We found that the chromomeres of the lampbrush chromosomes in the quail oocytes are very weakly Feulgen-positive or Feulgen-negative. Numerous small pyroninophilic nucleoli can be seen at the beginning of the lampbrush chromosome stage after staining with Unna. Pyroninophilia can also be demonstrated over part of the lateral loops, and, on the chromosomal axis, local pyroninophilic accumulations are obvious. The nucleoplasm contains very numerous small pyroninophilic granules which seem to be localized between the meshes of a very delicate network. All these pyroninophilic structures are no longer visible after enzymic digestion of the sections with RNase.

B. Second part of the lampbrush chromosome stage. When the germinal vesicle begins its migration to the periphery, the chromosomes shorten. The small nucleoli disappear. At the end of this stage the germinal vesicle still surrounded by yolk vacuoles comes to lie just below the basophilic cortical layer which has developed some time before. Some of the chromosomes still have a partly lampbrush configuration, others are present in the form of fine, short, straight treads (pyroninophilic after Unna) on which some spherules may be visible. The nucleoplasm is still filled with innumerable very small granules (pinkish after Unna). The germinal vesicle has still an ovoid or spherical form (diameter 160-180 μ m) and has now reached its maximum volume. During its evolution from early prelampbrush to late lampbrush chromosome stage, the diameter of the germinal vesicle increases some seven times, thus its increase in volume must be about 350 times.

This third stage, which follows the lampbrush chromosome stage, we have called the postlampbrush chromosome stage of meiosis. When the nucleus begins its penetration into the deepest basophilic cortical layer (in oocytes with a diameter of approximately 1·5 mm), two important simultaneous phenomena occur: (1) the sudden disappearance of the lampbrush chromosomes and the appearance of spherules in the most central part of this nucleus; (2) the increase in basophilia (RNase-sensitive) in the surrounding deep cortical layer, which then forms a cap over the nucleus (Fig. 1). At the moment when the nuclear cap has formed the cicatricular region is easily recognizable at the surface of the oocyte. At this moment also the dorsoventral axis of the future quail embryo is established (the dorsal side being situated at the surface of the cicatricular region, the ventral side towards the deeper part). At the beginning of this stage, during a relatively short period of time, a limited number of small spherules are seen spread irregularly in the nucleoplasm. Very soon, however, these spherules migrate to the most central part of the germinal vesicle. These central nuclear spherules have been shown to be Feulgen-positive (Callebaut, 1970b). The diameter of the cross-sectional area of these small Feulgen-positive, rounded bodies varies approximately 0·5-3 μ m, and vacuoles can usually be seen at their periphery (Fig. 2). They are usually found in two successive sections of the germinal vesicle only. Their exact number could not have been determined, but in favourable cases, when almost the whole group is localized in one section, we were able to distinguish up to 22 of these organelles (Fig. 3). These spherical, Feulgen-positive bodies are a characteristic feature of immature ovules with a germinal disc. They are only found in oocytes with a diameter of approximately 1·5-19 mm. Other Feulgen-positive structures cannot be demonstrated in the germinal vesicle during this stage. After staining with fast green, the localization of the central spherules can be seen with low-power magnification as lacunae in the densely green coloured nucleoplasm. The peripheral rim of nucleoplasm is, however, paler than the enormous central core. Under high-power magnification, hollow spheres bounded by a very delicate fast-green-colourable shell are seen in these centrally localized lacunae (Fig. 4). These hollow spheres correspond exactly to the unstained central Feulgen-positive spherules, and their shell is surrounded by a clear, often interrupted halo from which clear vesicular protrusions extend into the surrounding nucleoplasm. At high-power magnification, the whole nucleoplasm is seen to have a very fine mesh-like appearance. At the periphery of the group of central spherules some densely green, even smaller granules can often be seen in variable numbers after fast green staining. After the Unna stain, the central spherules have a green colour and are visible as such during the whole postlampbrush chromosome stage. The nucleoplasm after the latter stain presents a delicate, very weakly pyroninophilic network, and in some of the largest oocytes, very small pyroninophilic clumps can be discerned between and around the somewhat larger methylgreen-colourable central spherules. These small pyroninophilic organelles are, however, not always present. On rough stereometric calculations, as the volume of the spherical or ovoid germinal vesicles at the end of their lampbrush chromosome stage is approximately equal to the volume of the flattened coneshaped germinal vesicles at the end of the postlampbrush chromosome stage, we may assume that no significant alteration in volume takes place during the postlampbrush chromosome stage.

The postlampbrush chromosome stage is normally followed by the onset of maturation. Among the largest oocytes (diameter 17-19 mm) we occasionally found some in which the germinal vesicle wall was disintegrated. At the exact centre of the disintegrated germinal vesicle, chromosomes (often V shaped) or transition forms from central spherules to chromosomes (coloured green after Unna) are found.

In the animals of the third group (poorly illuminated) the diameter of the follicles does not exceed 1 mm, the whole stock of oocytes in these ovaries being in a developmental stage, corresponding to the prelampbrush or lampbrush chromosome stage of meiosis found in the regularly laying quails.

However, after Unna staining, the lampbrush chromosomes and lateral loops are seen to colour red much more intensely than in the case of quails exposed to continuous light. Oocytes in the postlampbrush chromosome stage cannot be found in Japanese quails exposed to poor illumination for long periods. A normally progressing postlampbrush stage is found only in the ovaries of quails exposed to continual or long-day illumination. The temperature of the environment does not greatly influence this stage since we could obtain regular egg laying equally well at temperatures of 0-5°C as at temperatures of 20-25°C.

Thus we could not confirm the observation of Fargeix (1964), who claims that egg production ceases in the Japanese quail at temperatures below 15°C.

The time spent by a normally developing oocyte in the postlampbrush stage must be approximately 3 weeks since adult quails of the poorly illuminated group (group 3) when exposed to continual illumination will start egg production after this period of time.

Thus the duration of the lampbrush chromosome stage can only be estimated to have an approximate maximum duration of 3 weeks, since normally a 6-weekold quail may begin egg production and at hatching, formation of intrafollicular oocytes (with a prelampbrush configuration) has already begun. The life-time of the early prelampbrush stage must be very variable: from a few days in first laid eggs to months or years for later ones.

On the autoradiographs of sections of oocytes in the adult animals of group 2 (treated with [³H]thymidine during their embryological life) and killed 50-80 days later during their egg laying period, the three stages of development found during the previously described cytological investigations can easily be recognized. In these oocytes, labelling can be found only on the chromosomes or central spherules.

(1) During the prelampbrush chromosome stage: labelling over the chromosomes can be found in most germinal vesicles at this stage. This labelling, however, does not always show a uniform distribution; some sections of a particular germinal vesicle being entirely devoid of grai ns, while others of the same germinal vesicle are clearly labelled on the chromosomes.

(2) During the lampbrush chromosome stage: owing to the enormous extension of the chromosomes in the now much more voluminous germinal vesicle, the number of grains per nuclear surface unit decreases sharply and may reach background level. When the grains overlying the chromosomes are sparsely scattered, it may be difficult or even impossible to conclude whether they are labelled or not.

(3) Most oocytes in the postlampbrush chromosome stage are found to be labelled on the Feulgen-positive central spherules (Fig. 5).

On the autoradiographs of oocytes obtained after [³H]thymidine injections in adult, regularly laying Japanese quails (group 1) we never observed any labelling of the chromosomes, central spherules or nucleoplasm. This was also the case for the oocytes of the animals in group 3 (poorly illuminated). By contrast, the paranuclear Balbiani yolk-body complex, so characteristic of the prelampbrush chromosome stage, is always diffusely but clearly labelled 2 h to several days after such an injection (Fig. 6). During the postlampbrush chromosome stage, labelling can only be observed over the subcortical cytoplasmic organelles from 1 h to several days after the injection of [³H]thymidine (Fig. 7).

After intraperitoneal [³H]uridine injection of the quails of group 1, intense and rapid (after only 30 min) RNase-sensitive labelling was found on the chromosomes at the prelampbrush chromosome stage. After 30 min we could also see labelling of the chromosomes at the lampbrush chromosome stage. This labelling was first confined to the chromosomes only, but soon (after 1 h or longer) the labelling was also found diffusely spread over the nucleoplasm. The resolution of our autoradiographic procedure, however, did not enable us to determine whether this labelling is due to incorporation of [³H]uridine into the nucleoplasm in situ (for instance, in the innumerable very small pyroninophilic granules) or if this label comes from rapidly synthesized RNA in the lampbrush chromosomes, which then moves promptly into the nucleoplasm. No appreciable labelling (even several hours or days after the injection) was found on the central spherules of the germinal vesicle during the postlampbrush chromosome stage. Usually no silver grains were visible over the nucleoplasm of oocytes at this stage.

In the third animal group (poorly illuminated), where the oocytes contain prelampbrush or lampbrush chromosomes only, the RNase-sensitive labelling over these chromosomes shows a pattern identical to that of group 1. Among the limited number of mature oocytes we have investigated during our experiments, only one was found 3 h after the intraperitoneal injection of [³H]uridine. No labelling was discerned over the chromosomes after autoradiography in this case.

Treatment during embryological life with successive applications of [³H]-thymidine in ovo, with the aim of labelling as high a number as possible of germinal vesicles of the final stock in adult quails, was based on our observations (Callebaut, 1968) that the first leptotene figures of meiosis may be found in the central (most advanced) part of the ovary of 10-day-old quail embryos. Thus the premeiotic DNA synthesis in the latter oocytes must occur some hours before. We therefore started our treatment with [³H]thymidine in 9·5-day-old quail embryos. Since the primary oocytes labelled by this procedure no longer divide, the [³H]thymidine incorporated in their chromosomal DNA will no longer be diluted by new DNA synthesis. However, part of rhe germ cells may have been labelled during their premitotic S phase (oogonia) and not during their premeiotic DNA synthesis, since at the periphery of the ovarian cortex oogonia continue to divide during the period of application of [³H]thymidine. This in ovo-labelled chromosomal DNA must be very stable since labelling of the chromosomes of the oocytes in such treated animals could still be demonstrated 80 days after hatching.

Our present study also clearly demonstrates that the central spherules, found in the germinal vesicle during the postlampbrush chromosome stage, represent contracted chromosomes because: (1) they are Feulgen-positive, (2) on the chromosomes at the end of their lampbrush stage, localized spherical accumulations resembling the central spherules may be found; (3) on the autoradiographs of the laying quails treated during their embryological life with [³H]thymidine pulses, a concentration of silver grains can be discerned over and around these central spherules; (4) during the onset of maturation we could observe transitional forms between the postlampbrush chromosomes and chromosomes of the first maturation spindle.

Our results show that the largest oocytes found in an adult Japanese quail, which after fecundation will form the quail embryos, develop from oogonia or oocytes already formed during her embryological life.

There occurs intense incorporation of [³H]uridine over the chromosomes during the prelampbrush chromosome.

This labelling seems to be due to macromolecular RNA since RNase (DNase free) treatment removes almost all the radioactivity from the nucleus. The diffuse labelling over the paranuclear Balbiani yolk-body complex at the prelampbrush chromosome stage after intraperitoneal injection of [³H]thymidine probably indicates mitochondrial DNA synthesis since the most voluminous part of the paranuclear Balbiani yolk-body complex consists of mitochondria. During the lampbrush stage there is both biochemical (RNase-sensitive incorporation of [³H]uridine) and cytochemical (RNase-sensitive pyroninophilia of the chromosomes) evidence of RNA synthesis. By contrast, during the postlampbrush chromosome stage our investigations could not afford any proof of the presence or synthesis of RNA in the central spherules. Transplantation experiments in enucleated amphibian eggs suggest that the cytoplasmic environment in the egg and in the pregastrulation embryo contains a regulatory factor for ribosomal RNA synthesis (Crippa, 1970). During the postlampbrush chromosome stage here described in the Japanese quail there is also autoradiographic and cytological evidence of arrested or very limited RNA synthesis in the germinal vesicle. Also, after a relatively enormous increase in volume of the germinal vesicle (several hundred times that of the original) during the lampbrush chromosome stage, its volume remains approximately constant during the ensuing postlampbrush chromosome stage. By contrast, during the latter period, part of the fundamental ooplasm (the radially placed basophilic subcortical cytoplasmic organelles and the basophilic cortical layer from which they arise) shows nucleic acid activity (Callebaut, 1970a,b, 1971, 1972a, b).

Thus there seems to occur a shift in activity from the germinal vesicle to the fundamental part of the ooplasm. It is remarkable that the sudden striking change in the chromosomal apparatus from a very active lampbrush stage to an inactive postlampbrush stage exactly corresponds to the ‘natural’ transplantation of the germinal vesicle from the central yolk-mass into the overlying basophilic cortical layer. The clear, irregular halo (unstainable by any of the stains used) which surrounds the spherical chromosomes, could be an artificial retraction cavity due to fixation. If, however, this halo really exists in the living oocyte, it may represent the materialization of the repression state in which the chromosomes are found during the postlampbrush chromosome stage.

La continuité dans les différents stades de développement des oocytes de la caille japonaise pondeuse a pu être démontrée grâce au marquage de leurs noyaux par applications successives de thymidine tritiée pendant la période intraembryonnaire préméiotique. Avant leur maturation les oocytes primaires intraovariens de la caille japonaise adulte traversent successivement trois stades importants. Dans chacun de ces stades les chromosomes ont une morphologie et un comportement cytochimique spécifique. Pendant le premier stade ou stade ‘prelampbrush’ les chromosomes sont Feulgen-positifs et se colorent en vert par la coloration au vert de méthyle-pyronine. Une incorporation d’uridine tritiée peut être observée à leur niveau après injection de ce précurseur radioactif par voie intrapéritonéale. Au début de cette période de durée très variable l’oocyte semble être dans un état de stabilité structurelle. L’existence dans l’ooplasme d’un très volumineux complexe paranucléaire (avec noyau vitellin de Balbiani) est caractéristique pour ce stade. Ce complexe peut être marqué après injection intrapéritonéale de thymidine tritiée à l’animal. Au second stade ou stade ‘lampbrush’ cette dernière structure disparaît et la réaction nucléale de Feulgen devient très faible ou disparaît au niveau des chromosomes. Après coloration au vert de méthyle-pyronine une pyroninophilie (sensible à la RNase) est visible au niveau des chromosomes plumeux et de leurs boucles. Après injection intrapéritonéale d’uridine tritiée, on trouve une incorporation rapide de ce précurseur dans les chromosomes plumeux et dans le nucléoplasme. Le marquage disparaît après digestion des coupes à la RNase. Pendant ce stade il y a donc évidence cytochimique et autoradiographique de synthèse de RNA dans la vésicule germinative. Pendant le troisième stade ou stade ‘postlampbrush’ l’activité dans la vésicule germinative diminue fortement; son volume reste constant et les chromosomes inactifs se retrouvent sous forme de sphérules centrales Feulgen-positives. Après injection intrapéritonéale d’uridine tritiée à la caille pondeuse on ne peut mettre en évidence sur les autoradiographies une incorporation de ce précurseur ni dans les chromosomes ni dans le nucléoplasme. Par contre, à ce moment au niveau des organites cytoplasmiques subcorticaux (qui se sont développés à partir d’une partie corticale del’ooplasme)on peut déceler la présence et/ou lasynthèse d’acides nucléiques. Le stade postlampbrush est caractéristique des oocytes immatures porteurs d’un disque germinatif et ne s’observe que chez des animaux en période de ponte régulière. Ce n’est pas la température ambiante, mais la durée de l’illumination journalière qui apparaît comme le facteur essentiel pour obtenir une ponte régulière chez la caille japonaise.

The author is very grateful to Professor L. Vakaet, Laboratory of Anatomy and Embryology, R.U.C.A., for his valuable criticisms.

The author also wishes to thank Mr G. Van den Broeck and Mr J. Swinnen for their skilful technical assistance, Mrs S. Bleyenbergh for typing the manuscript and last but not least Dr Hugh Crispin for reading and discussing the final text.