J. Embryol. exp. Morph. Vol. 20, 2, pp. 169-74, September 1968
With 1 plate
Printed in Great Britain
169
[3H]Uridine incorporation
during previtellogenesis and early vitellogenesis in
the oocytes of the chick (Gallus gallus)
By MARC CALLEBAUT 1
Radiobiological Department, Nuclear Energy Study Centre, Mol, Belgium
During the weeks following hatching of poultry, oocyte differentiation is most
advanced in the central part of the ovarian cortex. For instance, in the 5-day-old
chicken, oocytes surrounded by a ring of cuboidal cells (constituting early
follicles) are appearing in the central part of the cortex. At the extreme periphery
of the same cortex one occasionally can find a few germ cells which are still in
the late preleptotene, and often all the intermediate stages can be seen at intermediate localizations. D'Hollander (1904) has described the different types of
oocytes that can be found during the days following hatching. His investigations
indicate that during the so-called extrafollicular phase the nucleus probably
passes out of pachytene and into a stage when the chromatin is found in heavy
filaments split longitudinally in places. Later the cells go through a pseudoreticular stage in which the nucleoli and the nuclear membrane have reappeared.
Between the fifth and the twentieth day after hatching, the intrafollicular phase
sets in and the chromatin may also be found in the form of chains (chiasmata),
clearly indicating the diplotene stage. From this stage it passes through a
granular stage and becomes progressively retracted from the nuclear wall,
finally giving 'barbed' or lampbrush chromosomes. In the 20-day-old chick,
numerous oocyte nuclei with a lampbrush configuration and a large nucleolus
can be seen.
Greenfield (1966), in the course of her study of chick oocytes by electron
microscopy, discerned in the youngest intrafollicular oocytes a dispersion of the
Balbiani nucleus. The Golgi apparatus is no longer a single compact mass but
small regions of it are found near the nucleus, and mitochondria are scattered
throughout the ooplasm. The period of previtellogenesis in oocytes is essentially
a period of synthesis of protoplasm. The growth of the oocyte depends on the
increase and the evolution of cytoplasmic organelles. Vitellus is only formed
in very small amounts or not at all. For these reasons it seemed interesting to
investigate the incorporation of [3H]uridine during this period.
Incorporation of labelled precursors into the RNA of developing oocytes has
1
Author's address: Laboratories du C.E.N. a Mol-Donk, Mol, Belgium.
170
M. CALLEBAUT
been studied thoroughly in amphibians by Gall & Callan (1962), Davidson,
Allfrey & Mirsky (1964), and Brown & Littna (1964a, b). RNA synthesis
during the development of the oocytes of the sea urchin has been examined by
Gross, Malkin & Hubbard (1965).
MATERIAL AND METHODS
The experiments were performed on female chicks with sex-linked heredity
{Fx generation; Rhode Island Red x White Plymouth Rock).
Fifteen 6-day-old chicks received a single subcutaneous injection of 250 fie
of [3H]uridine. Ten chicks of 16, ten of 20, ten of 26 andfiveof 50 days received
a subcutaneous injection of 500/*c. Uridine-5-T from the Radiochemical
Centre, Amersham, England (TRA 178, batch 10, 3 Ci/mM), was used.
Two 16-day-old chicks received a single subcutaneous injection of 500 /ic
[3H]thymidine (specific radioactivity: 14 Ci/mM). At different times (see results)
after the injection chicks were killed and the ovaries removed and fixed in
acetic alcohol (1:3, v/v) at 4 °C for 18 h, embedded in paraffin and sectioned
at 5 JLL. After deparaffination and rehydration, some of the sections were treated
with RNAase or with DNAase.
For RNAase treatment, the sections were immersed for 1 h in Tris buffer at
pH 7-5 containing 0-2 mg RNAase/ml. For DNAase treatment, they were
immersed for 3 h at 37 °C in Tris buffer at pH 7-5 containing 0-1 mg DNAase/
ml and 00033 M MgCl2. Acid-soluble precursors were extracted from some of the
sections (treated or not with RNAase or DNAase) by treatment with 3 %
perchloric acid (PCA) at 4 °C for 20 min. After such treatment the RNAprecursor pools, soluble RNA and some messenger RNA, being water-soluble,
no longer register autoradiographically.
The slides are coated with nuclear emulsion L 4 (Ilford, England) by the
dipping method. After 2-4 weeks exposure in the dark and photographic
development the sections were stained with Groat's iron haematoxylin and
eosin.
RESULTS
1. After injection of [3H]uridine
1. Chicks injected 6 days after hatching and:
(a) Fixed 5 h later. Sections were treated with PCA. The label is present over
the nuclei of the most advanced (intrafollicular) oocytes localized in the central
part of the ovarian cortex. There is also labelling over some pseudoreticular
nuclei; but over the oocytes with nuclei containing heavy filaments of chromatin,
localized at the periphery of the cortex, no label can be seen. An exact count of
the number of grains over the cytoplasm was impossible since the ooplasm
was too small.
(b) Fixed 1 day later. Sections were treated with PCA. The label over the
oocytes was similar to that found after 5 h.
J. Embryol. exp. Morph., Vol. 20, Part 2
PLATE 1
Fig. 1. Autoradiograph of an oocyte in the ovary of a 16-day-old chick, 5 h after a subcutaneous injection of [3H]uridine (section treated with PCA). The nucleolus and the nucleus
are labelled but there is no appreciable label over the cytoplasm, x 1050.
Fig. 2. Autoradiograph of an oocyte of a 15-day-old chick, 9 days after a subcutaneous
injection of [3H]uridine (section treated with PCA). Note the very feeble nuclear labelling and
the intense cytoplasmic label, x 1050.
Fig. 3. Oocyte of a 6-day-old chick, starting intrafollicular development. The clear zone,
surrounded by flat cells, is directed toward the ovarian surface while the dense eosinophilic
part of the ooplasm, surrounded by young follicle cells, is localized on the opposite pole,
x1050.
M.CALLEBAUT
facing p. 171
Oocyte uridine incorporation
171
(c) Fixed 9 days later. Most of the oocytes are now intrafollicular. Sections
were treated with PCA. The label over the oocyte nuclei has decreased but
the ooplasm of all the intrafollicular oocytes is now intensely labelled (Plate 1,
fig. 2).
(d) Fixed 20 days later. Only very little label is still visible over the ooplasm.
(e) Fixed 1 month later. The label has entirely disappeared.
2. Chicks injected 16 days after hatching and:
(a) Fixed 5 h later. After treatment with PCA nearly all the oocyte nuclei are
intensely labelled. Almost always grains are found over or at the periphery of
the nucleolus (Plate 1, fig. 1). Nuclei of extrafollicular oocytes are not or only
weakly labelled.
Without PCA treatment the nuclei and the cytoplasm are more labelled than
after PCA treatment.
With DNAase digestion followed by PCA treatment most of the radioactivity
remained.
(b) Fixed 1 day later. After PCA treatment the label is now also found in the
cytoplasm. With DNAase digestion followed by PCA treatment most of the
radioactivity remained.
(c) Fixed 4 days later. After treatment with PCA the nuclear labelling in the
oocytes has decreased and the whole cytoplasmic surface is equally labelled, but
in some oocytes there is no or very feeble labelling over the central part of the
eosinophilic cytoplasmic area.
(d) Fixed 8 days later. After treatment with PCA the nuclear labelling of the
oocytes has decreased but the cytoplasm is heavily labelled.
(e) Fixed 2 months later. After PCA treatment no appreciable radioactivity
was found.
3. Chicks injected 20 days after hatching
We found the same results as after an injection at 16 days.
4. Chicks injected 26 days after hatching
The same pattern of incorporation was found as after the injection at 16 or
20 days but the radioactivity was less pronounced.
5. Chicks injected 50 days after hatching
Five hours later in some oocyte nuclei a few grains are found but cytoplasmic
labelling during the following days is insignificant. In general after [3H]uridine
injection practically all radioactivity is removed from the oocytes by a treatment
with RNAase and PCA.
II. After injection of [3H]thymidine
Sixteen days after hatching and with fixation 5 h or 5 days later, no appreciable
radioactivity is found over the oocytes but some of the follicle cells are labelled.
172
M. CALLEBAUT
DISCUSSION
The label found on our autoradiographs after PCA treatment is due to
macromolecular RNA and not to DNA since:
(1) RNAase treatment removes almost all the radioactivity from the oocytes.
(2) The tracer used in our experiments is Uridine-5-3H, which cannot be
incorporated into DNA, as thymidylic acid, unless the tritium label is replaced
with a methyl group.
(3) No incorporation of [3H]thymidine in the oocytes of the 16-day-old
chicks could be discerned, indicating the absence of DNA synthesis.
Thus the present research indicates clearly that during previtellogenesis uridine is actively incorporated into the RNA of the oocytes of the chick. This
incorporation may indicate net synthesis of new RNAs or only exchange (turnover) of already synthesized RNA. The kinetics of accumulation of the labelled
RNA in the nucleus and cytoplasm appear similar to that found by Gall &
Callan (1962) during their investigations on the incorporation of 3H-uridine in
the lampbrush chromosome of Triturus oocytes, but the whole phenomenon is
more accelerated in the chick since cytoplasmic labelling appears already 1 day
after injection of [3H]uridine. The incorporation of [3H]uridine in chick oocytes
appears to be much slower than in isolated embryonic mouse somatic cells
cultured in vitro (Monesi & Crippa, 1964). Just before the oocyte becomes wholly
intrafollicular and during early intrafollicular development (Plate 1, fig. 3) a
polarization of the ooplasm occurs, the cytoplasmic sector presenting an empty
or vesicular structure directed toward the surface of the ovary whereas the dense
eosinophilic sector, surrounded by well-developed follicle cells, lies on the diametrically opposite pole. Bartelmez (1912) found in the pigeon that the bilateral
organization of the egg and the position of the future embryo's long axis are
already determined early in the period of slow growth. This may therefore
indicate that during this early growth period a partial preformation of the
future embryo has taken place, probably through the mediation of RNAs
synthesized at this time. If there is a real synthesis of RNA at this stage, then
RNAs responsible for the synthesis of enzymes necessary for yolk formation
during the subsequent vitellogenesis also may be synthesized, since [3H]uridine
incorporation during early vitellogenesis (50 days) seems rather low. However,
we know nothing about the penetration of [3H]uridine through the surrounding
cells (for instance, the follicle cells) and therefore it is possible that the availability of uridine to the oocyte changes with time.
Since during the days following the injection of [3H]uridine there is a very
pronounced increase in the total number of grains over the intrafollicular
oocytes, there must be a [3H]uridine metabolite pool. This metabolite pool is
probably localized within the ooplasm. Indeed, 5 h after the injection of [3H]uridine to macromolecular intracytoplasmic RNA (after PCA treatment of the
sections) is found, but acid-soluble [3H]uridine compounds (omitting PCA
Oocyte undine incorporation
173
treatment) can be demonstrated. However, the clear-cut demonstration of a
uridine metabolite pool in the cytoplasm of the oocyte can probably only be
given by autoradiography of soluble labelled compounds. Indeed, the existence
of separate nucleotide pools in the nucleus and the cytoplasm with different
rates of equilibration with the added [3H]uridine (Harris, 1959) is also possible.
Uridine pool formation or RNA synthesis in the follicle cells or the follicleprecursor cells with subsequent translocation to the oocyte is another possibility.
The uridine metabolite pool must be present already before the oocytes are
intrafollicular since in the 6-day-old chick only a few intrafollicular oocytes are
present whilst the bulk of the oocytes are still in less advanced extrafollicular
stages. Nine days later, however, the majority of these oocytes are intrafollicular
and are intensely labelled. It is possible that the existence of a large uridine pool
is a characteristic of oocytes during their early development. This property
would offer the possibility of labelling nearly the whole 'stock' of germ cells
by a single injection during the period of previtellogenesis.
Contrary to the situation found with [3H]uridine the labelling of all the germ
cells with tritiated thymidine (for instance, during premeiosis) requires successive
pulses (Callebaut, 1967 a, b).
Our investigations could be of interest in research into mutation since a
highly significant increase of mutation frequency has been found in Drosophila
melanogaster spermatocytes after [3H]uridine administration, during late
[3H]uridine incorporation (Olivieri & Olivieri, 1965).
SUMMARY
Chicks of 6,16, 20, 26 (previtellogenesis) and 50 (vitellogenesis) days received
a single injection of [3H]uridine. Their ovaries were then fixed at different times
and the kinetics of accumulation of labelled RNA in the nucleus and cytoplasm
of the oocytes was investigated by autoradiography. The existence of an extensive intracytoplasmic uridine metabolite pool has been postulated in order to
explain the progressive increase in the number of grains over the ooplasm. After
a single [3H]uridine injection during the period of previtellogenesis it is possible
to label nearly the whole 'stock' of intrafollicular oocytes. One to two months
later, however, the radioactivity has entirely disappeared.
RESUME
Des jeunes poulets ages de 6, 16, 20, 26 (previtellogenese) et de 50 (vitellogenese) jours recoivent une seule injection d'uridine tritiee. Leurs ovaires ont
ete fixes a differents moments apres cette injection et l'accumulation du RNA
marque dans le noyau et le cytoplasme des oocytes a ete suivie par une etude
autoradiographique. L'existence d'une reserve intracytoplasmique d'uridine a
ete proposee a fin d'expliquer l'augmentation progressive du nombre de grains
12
JE EM 20
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M. CALLEBAUT
d'argent au-dessus de l'ooplasme. Avec une seule injection d'uridine tritiee il
est possible de marquer la presque-totalite des oocytes intrafolliculaires. Un a
deux mois plus tard la radioactivite a entierement disparue.
This work was supported by a grant from the Fonds de la Recherche scientifique fondamentale collective.
The author is grateful to Professor J. Brachet of the University of Brussels and to Dr
L. Ledoux, Department of Radiobiology, Nuclear Energy Study Centre of Mol, for their
valuable criticisms.
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(Manuscript received 25 January 1968)