/. Embryol. exp. Morph. Vol. 29, 1, pp. 145-157, 1973
Printed in Great Britain
145
Correlation between germinal vesicle
and oocyte development in the adult Japanese quail
{Coturnix coturnix japonica).
A cytochemical and autoradiographic study
By MARC CALLEBAUT 1
From the Laboratory of Anatomy and Embryology (Professor L. Vakaet),
R.U.C.A., Antwerp
SUMMARY
Continuity in the various developmental stages of the oocytes in the adult laying Japanese
quail may be demonstrated with a method of whole stock labelling of their nuclei by successive
applications of [3H]thymidine during their premeiotic period.
Before they mature, the intraovarian oocytes of the adult Japanese quail go through three
important successive stages. In each of these stages the chromosomes have a distinct morphology and cytochemical behaviour. During the first stage or prelampbrush chromosome stage,
the chromosomes are Feulgen-positive or green after Unna, and on the autoradiographs
show intense incorporation of [3H]uridine after injection of this RNA precursor into the
animal. During the beginning of this period, which is of very variable duration, the oocyte
seems to be in a state of structural stability. The most prominent feature found in the
ooplasm of oocytes at this stage is the very large paranuclear Balbiani yolk-body complex,
which can be found labelled after injection of [3H]thymidine into the animal. During the
second stage or lampbrush chromosome stage, the Feulgen nuclear reaction in the chromosomes weakens or becomes negative and the paranuclear Balbiani yolk-body complex disappears. After Unna, the lampbrush chromosomes and their lateral loops are seen to be
pyroninophilic.
On the autoradiographs after intraperitoneal injection of [3H]uridine rapid and intense
RNase-sensitive incorporation of this precursor over the chromosomes and nucleoplasm may
be noted. During this stage there is thus both cytochemical and autoradiographic evidence
for RNA synthesis in the rapidly enlarging germinal vesicle. During the third or postlampbrush
stage activity in the germinal vesicle sharply decreases; the volume of the germinal vesicle
no longer increases, the very contracted chromosomes are present in the form of Feulgenpositive vacuolized central spherules, and, after intraperitoneal injection of [3H]uridine into
the adult laying quail, incorporation cannot be demonstrated in the chromosomes. By contrast, the fundamental part of the ooplasm derived from the cortex, at that moment shows
both cytochemical and autoradiographic evidence of the presence and/or synthesis of nucleic
acids. The postlampbrush stage is a characteristic feature of non-mature oocytes with a
germinal disc and can only be found in regularly laying Japanese quails exposed to full daytime or continuous illumination.
1
Author's address: Laboratory of Anatomy & Embryology, Rijksuniversitair Centrum,
Groenenborgerlaan, 171, 2020-Antwerp, Belgium.
E M B 29
146
M. CALLEBAUT
INTRODUCTION
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 'nucleoles nucleiniens'
because he found that they later constituted the chromosomes of the first
maturation spindle.
In previous work (Callebaut, 19706) 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.
MATERIALS AND METHODS
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 /.id of [3H]thymidine 6 (10 Ci/mM) in 50 /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).
Cytological study
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).
Germinal vesicle and oocyte development
147
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 ju,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
H-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.
Autoradiographic study
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 3-4 h. 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.
148
M. CALLEBAUT
RESULTS
A. Cytological study
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.
First stage
The smallest oocytes found in the ovary of adult Japanese quails having a
diameter from 30-50 jum are in this stage. They contain a spherical or ovoid
peripheral germinal vesicle with a diameter of 20-30 /*m. However, somewhat
larger oocytes with a diameter of up to approximately 250 /tm. and containing a
germinal vesicle with a diameter of 40-50 jam 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 /*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.
Second stage or lampbrush chromosome stage
This includes oocytes with a diameter varying from 250 to approx. 1000 jam.
During the first part of this stage (consisting of oocytes with a diameter from
250 to approx. 500 jam), 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
Germinal vesicle and oocyte development
149
(oocytes from approx. 500 to approx. 1000 jum 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/tm) 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.
Third stage
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: (I) the sudden disappearance of the lampbrush chromosomes and the
appearance of spherules in the most central part of this nucleus; (2) the increase
150
M. CALLEBAUT
2 '•"
*
Germinal vesicle and oocyte development
151
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, 19706).
The diameter of the cross-sectional area of these small Feulgen-positive, rounded
bodies varies approximately 0-5-3 fim, 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
FIGURES
1-7
Fig. 1. Perpendicular section of quail oocyte, diameter approximately 1-5 mm. The
germinal vesicle is penetrating the basophilic cortical layer. The latter now forms
a nuclear cap over the germinal vesicle. Note the disappearance of the lampbrush
chromosomes and the intense basophilia in the nuclear cap. At this moment the
germinal disc has just formed. (Unna stain.) Scale line = 100/tm.
Fig. 2. Section of the central part of the germinal vesicle of a Japanese quail oocyte,
diameter 8 mm, after Feulgen staining. The central Feulgen-positive vacuolized
spheres represent contracted post-lampbrush chromosomes. Scale line = 10 /tm.
Fig. 3. Perfectly tangential section of germinal disc of a 4-5 mm diameter oocyte. At
the centre of the apparently empty germinal vesicle, numerous chromosomes in the
postlampbrush stage can be seen. In the surrounding ooplasm, radially arranged
subcortical cytoplasmic organelles are obvious (iron haematoxylin and eosin). Scale
line = 100/tm.
Fig. 4. Tangential section of central part of germinal vesicle of 7-5 mm quail oocyte
at high magnification after fast green staining. In some of the lacunae, hollow
spheres bounded by a delicate fast-green-stainable shell are visible. These hollow
spheres correspond to the here unstained Feulgen-positive central spherules, and
their shell is surrounded by a clear halo from which vesicular transparent protrusions extend into the nucleoplasm. Scale line = 10 (im.
Fig. 5. Tangential section through the central part of the germinal vesicle of an
oocyte 14 mm in diameter from a regularly laying Japanese quail, treated //; ovo
with successive applications of [3H]thymidine. The central spherules (postlampbrush
chromosomes) are clearly labelled (from an autoradiograph stained with iron
heamatoxylin and eosin). Scale line = 20/tm.
Fig. 6. Small oocyte of an adult laying Japanese quail 2 h after an intraperitoneal
injection of 2 mCi of [3H]thymidine. Labelling is found over the paranuclear Balbiani
yolk-body complex (from an autoradiograph stained by iron haematoxylin and
eosin). Scale line = 50/tm.
Fig. 7. Autoradiograph of tangential section of quail oocyte, diameter 4-5 mm
(fixation 1 h after an intraperitoneal injection of 1 mCi [3H]thymidine). On the
right the unlabelled germinal vesicle is seen. In the ooplasm the labelled, subcortical
cytoplasmic organelles are visible (iron haematoxylin and eosin). Scale line = 50 /tm.
152
M. CALLEBAUT
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.
Maturation
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
Germinal vesicle and oocyte development
153
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.
Using [zH]thymidine
B. Autoradiographic study
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 grains, 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
154
M. CALLEBAUT
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 If 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).
Using [3H]uridine
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.
DISCUSSION
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]thym.idine incorporated in their chromosomal DNA will no longer be
diluted by new DNA synthesis. However, part of the germ cells may have been
labelled during their premitotic S phase (oogonia) and not during their pre-
Germinal vesicle and oocyte development
155
meiotic 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 (RNasesensitive 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, I972a,b).
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M. CALLEBAUT
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.
RESUME
Relation entre Vaspect de la vesicule germinative et le developpement
de Voocyte chez la caille japonaise
La continuite dans les differents stades de developpement des oocytes de la caille japonaise
pondeuse a pu etre demontree grace aii marquage de leurs noyaux par applications successives
de thymidine tritiee pendant la periode intraembryonnaire premeiotique. 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 specifique. Pendant le premier stade ou stade 'prelampbrush'
les chromosomes sont Feulgen-positifs et se colorent en vert par la coloration au vert de
methyle-pyronine. Une incorporation d'uridine tritiee peut etre observee a leur niveau apres
injection de ce precurseur radioactif par voie intraperitoneale. Au debut de cette periode
de duree tres variable l'oocyte semble etre dans un etat de stabilite structurelle. L'existence
dans l'ooplasme d'un tres volumineux complexe paranucleaire (avec noyau vitellin de
Balbiani) est caracteristique pour ce stade. Ce complexe peut etre marque apres injection
intraperitoneale de thymidine tritiee a l'animal. Au second stade ou stade 'lampbrush'
cette derniere structure disparait et la reaction nucleate de Feulgen devient tres faible ou
disparait au niveau des chromosomes. Apres coloration au vert de methyle-pyronine une
pyroninophilie (sensible a la RNase) est visible au niveau des chromosomes plumeux et de
leurs boucles. Apres injection intraperitoneale d'uridine tritiee, on trouve une incorporation
rapide de ce precurseur dans les chromosomes plumeux et dans le nucleoplasme. Le marquage
disparait apres digestion des coupes a la RNase. Pendant ce stade il y a done evidence cytochimique et autoradiographique de synthese de RNA dans la vesicule germinative. Pendant
le troisieme stade ou stade 'postlampbrush' l'activite dans la vesicule germinative diminue
fortement; son volume reste constant et les chromosomes inactifs se retrouvent sous forme
de spherules centrales Feulgen-positives. Apres injection intraperitoneale d'uridine tritiee
a la caille pondeuse on ne peut mettre en evidence sur les autoradiographies une incorporation
de ce precurseur ni dans les chromosomes ni dans le nucleoplasme. Par contre, a ce moment
au niveau des organites cytoplasmiques subcorticaux (qui se sont developpes a partir d'une
partie corticale de l'ooplasme) on peut deceler la presence et/ou lasynthese d'acides nucleiques.
Le stade postlampbrush est caracteristique des oocytes immatures porteurs d'un disque
germinatif et ne s'observe que chez des animaux en periode de ponte reguliere. Ce n'est pas
la temperature ambiante, mais la duree de l'illumination journaliere qui apparatt comme le
facteur essentiel pour obtenir une ponte reguliere 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.
Germinal vesicle and oocyte development
157
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(Manuscript received 12 May 1972, revised 26 June 1972)