/. Embryol. exp. Morph. Vol. 22, 2, pp. 229-51, September 1969
229
Printed in Great Britain
Observations on the origin of the ' germinal
cytoplasm' in Xenopus laevis
By RENATA CZOLOWSKA 1
From the Institute of Zoology, University of Warsaw, Poland,
and the Hubrecht Laboratory, Utrecht, Holland
The early appearance of the 'germinal cytoplasm' and its behaviour during
the formation of cells which are thought to represent the primordial germ cells
have been described in detail for Rana temporaria by Bounoure (1927, 1934,
1939) and Bladder (1958). These observations were extended to Xenopus laevis
(Nieuwkoop, 1956; Nieuwkoop & Faber, 1956; Blackler, 1958), Bufo bufo
(Blackler, 1958), Ranapipiens (Berardino, 1961), Discoglossus pictus (Gipouloux,
1962 a) and Rana esculenta (Hammer, cited by Blackler, 19656).
The above findings agree with respect to the earliest detection of the 'germinal
cytoplasm'. Its first appearance was noticed as early as in the fertilized, unsegmented egg, where small, distinctly staining islands of cytoplasm are localized
just under the cell membrane in an area around the vegetative pole of the egg.
During cleavage the 'germinal cytoplasm' is distributed between the vegetative blastomeres directly surrounding the vegetative pole. At the beginning of
gastrulation some cells containing 'germinal cytoplasm' are detectable in the
presumptive endoderm and are, from then on, considered as true primordial
germ cells, incapable of undergoing any somatic differentiation. They retain
their embryonic character with respect to yolk and pigment content and
relatively large size. Subsequently, the primordial germ cells are gradually
displaced in the endoderm by morphogenetic movements and afterwards,
presumably, by active migration until they reach a position corresponding to
that of the future germinal ridges.
Making use of the localization of the 'germinal cytoplasm', Bounoure
(1935a, b, 1937a, b, c, d, 1939, 1950), Aubry (1953) and Bounoure, Aubry &
Huck (1954) have demonstrated that irradiation of the vegetative hemisphere
of fertilized, unsegmented eggs of R. temporaria with ultraviolet light (u.v.) has a
very definite effect on the future germinal line. After such treatment the gonads
of fully metamorphosed frogs are reduced in size and possess a markedly
reduced number of gonocytes, causing partial or complete sterility. Padoa (1963,
1964) obtained similar results with R. esculenta. The effect of u.v. on the eggs
1
Author's address: Institute of Zoology, University of Warsaw, Warsaw 64, Poland.
230
R. CZOLOWSKA
of R.pipiens was extensively studied by Smith (1966). With a defined dose, wavelength and time of exposure he obtained complete sterilization in 100 % of the
larvae by irradiating the eggs from the vegetative side just before the first cleavage.
Smith was also able to show that the germinal line is restored if vegetative plasm
of a non-irradiated egg is injected into the vegetative half of an irradiated one.
These results allow us to conclude that a u.v.-sensitive material localized
around the vegetative pole of the fertilized egg is responsible for germ-line
differentiation.
Less clear results have been obtained with surgical methods. According to
Nieuwkoop & Suminski (1959) and Fischiarolo (1960) the removal of vegetative
material in dividing eggs (4-cell stage) or early embryos (blastulae) of Xenopus
laevis and Discoglossus pictus respectively does not influence the future germinal
line of the larvae. However, Monroy (1939) and Gipouloux (19626) reported
positive results after removing the endoderm from neurulae, and Librera (1964)
succeeded in obtaining sterile larvae when the cytoplasm lying nearest to the
vegetative pole had been removed from uncleaved eggs.
Special attention has been paid to two basic problems: (1) whether cells which
contain identifiable 'germinal cytoplasm' should be considered as true primordial germ cells, i.e. whether they are transformed into the true gametes
in the course of development, and (2) whether primordial germ cells represent
the exclusive source of all gametes. In both cases a positive answer seems
to be justified when the results of Blackler (1960, 1961, 1962a, b, 1965tf, b) and
Blackler & Fischberg (1961) are considered.
From morphological observations it is known that the 'germinal cytoplasm'
is crowded with mitochondria, rich in pigment granules and very often associated with small yolk platelets. Some RNA is present as was demonstrated by a
cytochemical test by Blackler (1958). This is consistent with the speculations of
Padoa (1964) and with the deduction of Smith (1966), who found that the range of
the u.v. spectrum within which the germinal line is damaged corresponds to that
within which nucleic acids (both RNA and DNA) absorb and hence are affected.
In the present study an attempt is made to elucidate the origin of the 'germinal
cytoplasm'.
MATERIAL AND METHODS
Fertilized eggs, unfertilized eggs, ovulated oocytes and ovarian oocytes of
Xenopus laevis were used in the course of these studies, in total about 250
specimens.
Fertilized and unfertilized eggs were obtained after injecting toads with
gonadotrophic hormone (Physex, Leo Pharmaceutical Products), the males with
40 i.u. on two successive days and the females with 200 i.u. on the second day.
Ovulated oocytes were taken from hormonally stimulated females which were
killed some time after they started to lay normal eggs. Material was collected
from the uterus, from different portions of the oviduct and from the body cavity.
Germinal cytoplasm in Xenopus
231
As a source of ovarian oocytes two kinds of females were used, those
stimulated by hormone injection and unstimulated ones. After dissection small
pieces of the ovary were fixed and individual oocytes were isolated with
forceps.
Oocytes obtained after ovulation was induced in vitro were an additional
source of material. For this purpose small ovarian fragments were suspended
in Ringer solution (pH 7-2) containing 25 and 50 i.u. of Physex/ml. The Ringer
solution used contained (g/1.): 6-5 g NaCl, 014 g KC1, 012gCaCJ 2 , 0-2 g
NaHCO 3 , 005 g NaH 2 PO 4 .H 2 O. After 8 h hormone treatment all oocytes
lying freely on the bottom of the vials were collected and placed in Ringer for
40 min or 13 h before fixation.
Short fixation (4-6 h) in neutral 4 % formol containing 0-5 % cetylpiridinium
chloride (the presence of cetylpiridinium chloride facilitates decapsulation) with
simultaneous decapsulation in the fixative was followed by standard dehydration through alcohol and embedding in hard paraffin (M.P. 58 °C). Storage for
48 h in isoamyl acetate, before embedding, was found to be helpful for obtaining
good sections.
Serial sections, 5-7 fi thick, were cut either parallel or perpendicular to the
animal-vegetative axis of the egg. The sections were stained using the Azan
method (Romeis, 1948), but omitting differentiation with aniline/alcohol and
treatment with phosphotungstic acid. Staining in warm azocarmine for 15-30 s
was followed by rinsing in distilled water and staining in orange G-aniline
blue for 30 min.
As a specific method for RNA detection, staining with methyl green-pyronine
was used. The original formula of Unna (1913) was modified according to
J. Brachet (personal communication). The staining mixture consisted of 015 g
of methyl green and 0-25 g of pyronine-Y dissolved in 100 ml of acetate buffer
(pH 4-7). Serial sections were mounted alternately on slides. One slide
from every sample was always treated with an aqueous solution of RNase
(Worthington Biochemical Corporation), at a concentration 0-2 mg/ml, for
1-2 h at 37-38 °C. As an additional control some slides were incubated in
distilled water only. Subsequently, all slides were stained together for 1-2 h in
the staining mixture. Short rinses in distilled water and 96 % alcohol were
followed by dehydration in absolute alcohol, clearing in xylol and mounting
in DePeX.
RESULTS
Ovarian oocytes
Large oocytes isolated from the ovary are surrounded by a triple sheet consisting of peritoneum, connective tissue and follicular epithelium (Wartenberg
& Schmidt, 1961; van Gansen, 1966). An underlying layer staining strongly with
aniline blue represents the vitelline membrane (Hope, Humphries & Bourne,
1963; Tokin & Gabayeva, 1963; Kemp & Istock, 1967).
232
R. CZOLOWSKA
Detailed descriptions of the amphibian oocyte with respect to yolk and pigment distribution, morphology of the nucleus and localization of the cytoplasm
have been given by Wittek (1952) and Kemp (1953, 1956).
An animal-vegetal 'gradient' in the distribution of the cytoplasm is well
marked (Fig. 1 A). In the whole animal hemisphere large quantities of blue-staining cytoplasm can be found. The pattern of its distribution is reminiscent of a
Germinal cytoplasm in Xenopus
y ^ r
..7
*i,
•
*
,F - • • • • • . • • . • • .
• * * " • V
!
j "
-I
233
•••*"
I ,
VP
500//
Fig. 2. Distribution of cytoplasm in a fully grown oocyte isolated from the ovary.
Meridional section. GV, Germinal vesicle; VP, vegetative pole region.
FIGURE 1
A. Meridional section of fully grown oocyte isolated from the ovary. Stained with
Azan method. An accumulation of cytoplasmic material (AC) is seen on the vegetative side of the germinal vesicle (G V). x 40.
B. Nucleus of ovarian oocyte, meridional section. Azan staining. Cytoplasmic
material (AC) accumulated underneath the germinal vesicle (GV). x 97.
C. Meridional section of vegetative pole region of ovarian oocyte under high
magnification. Stained with methyl green-pyronin. RNA-containing patches
(PAT) are seen among the yolk platelets (YP) at the periphery of the oocyte.
EV, Envelopes of the oocyte. x 910.
D. Meridional section of ovulated oocyte collected from the body cavity. Stained
with methyl green-pyronin. Germinal vesicle dissolved. Clear gradient of the
distribution of RNA with maximum in the animal hemisphere (AN). VEG, Vegetative hemisphere. x41.
E. Vegetative pole of an oocyte ovulated /// vitro. Meridional section, Azan staining.
Islands of darkly staining cytoplasm (ISL) are seen at the periphery of the oocyte.
x900.
F. As E, stained with methyl green-pyronin. V1T, Vitelline membrane; ISL, islands
of darkly staining cytoplasm, x 850.
234
R. CZOLOWSKA
thick, ramifying, three dimensional network. The centre of the oocyte is
occupied by a dense network of fine granular cytoplasm which seems to originate from the dispersion of material accumulated underneath the nucleus
(Fig. 1A, B). The amount of cytoplasm decreases markedly towards the
vegetative pole (Fig. 2).
The pigment around the vegetative pole forms a layer about 15-28 /-t thick;
its granules are much less numerous and larger in diameter than those of the
animal hemisphere.
PAT
SUB
INT
Fig. 3. Tangential section of the vegetative pole region of a fully grown oocyte
isolated from the ovary (distribution of the vegetative pigment not indicated).
Boundary between peripheral zone and follicular epithelium not clearly visible.
N, Nuclei of follicular epithelium and remaining envelopes; SUB, subperipheral
zone crowded with small yolk platelets; INT, internal zone laden with large yolk
platelets; PAT, patches of compact RNA-rich cytoplasm. Patches in the subperipheral zone indicated with fine dotting.
The area around the vegetative pole of the oocyte contains material which is
distributed in three distinct zones. The peripheral zone appears directly underneath the vitelline membrane as a thin cytoplasmic layer populated with small
yolk platelets. The underlying subperipheral zone is laden with small yolk
platelets which are packed very tightly together, forming a compact layer. The
cytoplasm occurs as delicate, dispersed patches. The internal mass is filled with
large yolk platelets, very rarely interspersed by small yolk platelets. The cytoplasm can be seen as compact patches situated among the platelets (Fig. 3).
Methyl green-pyronine staining shows an animal-vegetal 'gradient' of RNA
corresponding to the localization of the cytoplasm. Within this gradient the
area around the vegetative pole, although relatively poorer in RNA than the
animal hemisphere, nevertheless reacts strongly. It seems that some RNA-
Germinal cytoplasm in Xenopus
235
containing material accumulates there, also in the zone corresponding to the
vegetative pigment and the small yolk platelets (Fig. 1C).
No differences in any series were found in the fine structure of oocytes from
unstimulated as against hormonally induced females.
Oocytes collected from the body cavity
One of the characteristic features of ovulated oocytes is the presence of a single
envelope, the vitelline membrane.
The cytoplasm is distributed along an animal-vegetal 'gradient' (Fig. ID).
After the breakdown of the germinal vesicle the animal hemisphere appears to
be filled with fine granular cytoplasm and compact clusters of dark-staining
material, originating from the disintegrated nucleus. The vegetative hemisphere
seems much poorer in cytoplasm than the corresponding region of the nonovulated oocyte. The cytoplasm forms a very fine network, highly dispersed
among the yolk platelets.
PER
SUB
Fig. 4. Schematic drawing illustrating a part of the vegetative pole region of an
oocyte collected from the body cavity (vegetative pigment not indicated). PER,
Peripheral zone composed of the strip of cytoplasm and yolk platelets with changed
staining properties; SUB, subperipheral zone; ISL, cytoplasmic island crowded
with mitochondria.
In the vegetative pole region the three concentric zones of material are more
distinct. The peripheral zone, composed of the outermost strip of cytoplasm and
populated with small yolk platelets, is underlain by a subperipheral zone which
appears to be crowded mainly with small and partly with large yolk platelets
(Fig. 4). The internal mass, in sectioned material, is often separated from the
subperipheral zone by a crack (Fig. 1D). The crack itself is an artifact, but its
presence reflects true differences in composition and properties of both zones
which come to light during the histological procedure.
236
R. CZOLOWSKA
D
Germinal cytoplasm in Xenopus
237
At the periphery around the vegetative pole a striking change in the staining
properties of the yolk platelets occurs (Fig. 4). This consists of a loss of affinity
for orange G and simultaneous increased staining with aniline blue. In a single
platelet the change is seen, at first, at the outer margin and spreads subsequently
towards its centre. The material of the platelet seems to be gradually consumed,
a process leading finally to the disintegration of the platelet. The change in
staining is confined mainly to small yolk platelets in the peripheral and subperipheral zones, but some large platelets are also affected. The pattern of
distribution of the zone of altered yolk differs from oocyte to oocyte. In some
the change is visible over the whole vegetative periphery, up to the equator, but
other oocytes show it only in a much more limited area.
One could suppose that the changes represent degenerative phenomena
related to absorption of yolky material in overripe oocytes, as was demonstrated
by a safranin staining method by Selman & Pawsey (1965). In order to check
this possibility the proportion of successful fertilization obtained with eggs
coming from the same female as ovulated oocytes collected afterwards from the
body cavity was determined. The percentage of successfully cleaving eggs was
estimated as 80 %. In the light of this high percentage it is rather unlikely that
the ovulated oocytes studied were abnormal.
In many oocytes the region around the vegetative pole is marked by the
presence of blue cytoplasmic islands, distributed through the yolk (Fig. 5 A, B).
These islands have always been detected only in the most vegetative sector and
are localized exclusively within the peripheral and subperipheral zones. The
size of the islands varies from 3 to 15 [i and their number varies from oocyte to
oocyte. Under oil immersion their heavily stained cytoplasm is seen to be
crowded with oblong granules, which are slightly larger than the pigment
granules of the vegetative area (Fig. 4). In their size and appearance the granules
closely resemble mitochondria. The localization of the islands often varies
FIGURE 5
A. A part of the vegetative pole region of an ovulated oocyte collected from the
body cavity. Meridional section, Azan staining. Three large islands of cytoplasmic
material (ISL) are situated at the boundary between subperipheral (SUB) and
internal zones (INT), x 640.
B. Vegetative pole of an ovulated oocyteunder high magnification. Meridional section,
Azan staining. Two large cytoplasmic islands (ISL) localized at the boundary between
subperipheral (SUB) and internal zones (INT). VIT, Vitelline membrane, x 920.
C. As B, stained with methyl green-pyronin. x 950.
D. Tangential section of the vegetative pole region of an egg collected and fixed at
the time of insemination. Azan staining. Darkly staining cytoplasmic islands are distributed throughout subperipheral zone, x 760.
E. Tangential section of the vegetative pole region at the two-cell stage. Azan
staining. Islands of the 'germinal cytoplasm' (ISL) are distributed parallel to the
cleavage furrow. SP, Space separating both blastomeres. x 520.
238
R. CZOLOWSKA
within one and the same specimen. Some of them can be detected in very close
proximity to the cytoplasm of the peripheral zone. Others seem to be slightly
separated from the peripheral cytoplasm. Finally some are completely isolated
from the peripheral zone, lying deep among the yolk, at the boundary between
the subperipheral and the internal zones.
The general distribution of the pyronine-staining material corresponds to
that of the cytoplasm. The animal half appears to be extremely rich in RNA in
both the granular cytoplasm and the clusters. There is a very sharp decline in
amount of RNA in the cytoplasm filling the oocyte below the equator. The
vegetative pole region is, as a rule, very poor in RNA, and pink-staining
material forming a dispersed network in the yolk can barely be seen there.
Nevertheless, some sections show RNA-rich islands occupying the most vegetative region and corresponding in their position and appearance to the blue
cytoplasmic islands described above (Fig. 5C). Sections have also been found
which illustrate the closeness, of the RNA-rich islands to the peripheral cytoplasm.
Oocytes ovulated in vitro
Oocytes from two experimental groups have been used for preliminary
studies. Group I consists of oocytes which were collected after 8 h of hormone
treatment and kept for 40 min in Ringer solution before fixation. Group II
consists of oocytes collected after 8 h hormone administration and cultured
subsequently for 13 h in Ringer solution before fixation. Oocytes obtained in
these ways are always coated with a vitelline membrane.
The pattern of distribution of yolk and cytoplasm resembles that of oocytes
ovulated in vivo. Group I oocytes are characterized by an accumulation of cytoplasm in the centre of the upper hemisphere, while those of group II reveal a
more uniform distribution of material in the upper half of the egg. In both
groups granular cytoplasm and cytoplasmic clusters are always present.
In the region around the vegetative pole the yolky material of the subperipheral zone and the internal mass is apparently condensed in oocytes of group I,
but more loosely distributed in group II oocytes. The same is observed with
respect to pigment localization. The layer of vegetative pigment in oocytes of
group I is 15—19 /* thick and increases in group II to 18-56 /i.
The peculiar changes in staining properties of the yolk noticed in oocytes
obtained from the body cavity have never been observed in oocytes ovulated
in vitro.
Cytoplasmic islands around the vegetative pole are rather scarce and barely
discernible in group I oocytes, but they become clearly visible in the peripheral
and subperipheral zones of oocytes belonging to group II. Islands localized
in the peripheral zone are flattened and about 5—15 /^ long; they are continuous
with the peripheral cytoplasm and distributed at irregular intervals along the
whole vegetative side (Fig. 1E). In some regions islands can be found associated
Germinal cytoplasm in Xenopus
239
with small protrusions of the peripheral zone into the subperipheral layer.
Many other islands occupy a more internal position in the subperipheral zone
and even in the internal mass. An array of oblong granules seems to be a characteristic of the internal composition of these islands (Fig. 6).
Cytochemical tests made on samples from group II reveal a general animalvegetative gradient of distribution of RNA. In the region around the vegetative
pole numerous islands extremely rich in RNA occupy a position just underneath
the peripheral cytoplasm. RNA-rich islands occur also in the subperipheral
zone and even in the internal mass (Fig. 1F). After RNase treatment some of the
islands still retain a weak pinkish colour.
PER
VIT
SUB
25//
Fig. 6. Schematic drawing illustrating a part of the vegetative pole region of an
oocyte ovulated in vitro (vegetative pigment not indicated). VIT, Vitelline membrane; PER, peripheral zone; SUB, subperipheral zone; ISL, cytoplasmic islands
crowded with mitochondria and rich in RNA.
Oocytes collected from the oviduct
Oocytes from different parts of the oviduct were classified into four groups.
(I) Oocytes collected from the uppermost part of the tube, in the neighbourhood of the ostium, surrounded only by the vitelline membrane.
(II) Oocytes collected from the upper part of the tube, also surrounded only
by the vitelline membrane.
(III) Oocytes collected from the middle part of the tube, surrounded by the
vitelline membrane and partially by jelly.
(IV) Oocytes collected in the lowest part of the tube, near the uterus,
surrounded by the vitelline membrane and fully coated with jelly.
Except for group 1, all samples were cut tangentially to the vegetative pole.
In all the four groups studied a progressive change is observed in the peripheral zone near the vegetative pole, which becomes more compact and distinct
as oocytes are transported down the oviduct. Yolk platelets with altered staining
240
R. CZOLOWSKA
properties can only be found peripherally in the vegetative region of group I
and II oocytes (Fig. 7).
The subperipheral zone and internal mass are characterized by a rather compact appearance. Some variations are observed with respect to the distribution
of the large yolk platelets. In some cases they appear to be absent from the
peripheral and subperipheral zones, in others they approach quite closely to the
periphery of the oocyte.
SUB
Fig. 7. Schematic drawing illustrating a part of the vegetative pole region of an
oocyte collected from the uppermost part of the oviduct (vegetative pigment not
indicated). For the abbreviations see Fig. 4.
In oocytes of all four groups intensely stained bodies gradually appear in the
peripheral and subperipheral zones. These spots look like vacuoles and their
number, shape and size vary from sample to sample.
Some of the samples studied contain characteristic cytoplasmic islands (Fig. 7).
These have been found distributed within all three regions, in variable size and
number. The islands appear less regular in shape, more compact and more
heavily stained than corresponding ones from previous stages. Only very
detailed observation reveals their granular composition. Cytoplasmic islands
are tightly associated with small yolk platelets. Pigment granules associated
with the cytoplasmic islands are present in the same concentration as in the
surrounding regions.
Cytochemical tests made on samples from group II reveal the presence of
islands which are significantly richer in RNA than the very diffuse staining
regions of the peripheral cytoplasm at the vegetative side. The size and localization of the RNA-rich islands correspond to that of the blue cytoplasmic islands
observed after Azan staining.
Germinal cytoplasm in Xenopus
241
Eggs collected from the uterus and unfertilized eggs
If serial tangential sections of the vegetative pole region are studied, the three
zones are more or less clearly distinguishable. In all cases the peripheral zone
as well as the outermost region of the subperipheral zone are marked by the
presence of bodies, which are already observed in the previous stage, but have
now increased in number and size. These bodies very likely represent the
cortical vacuoles already described by many authors.
SUB
Fig. 8. Tangential sections of the vegetative pole region of unfertilized eggs (distribution of the vegetative pigment not indicated). A, Distinct separation of all three
zones. B, Obliterated pattern of yolk distribution. SUB, subperipheral zone; INT,
internal zone; ISL, islands of RNA-rich cytoplasm.
Variations are observed in the general distribution of the yolky material
which seem to be related to some unknown individual differences between eggs
coming from different females. According to the type of variations two main
groups of eggs can be distinguished.
The first group represents unfertilized eggs where a very distinct separation
of all three zones is observed (Fig. 8 A). The subperipheral zone is pronounced,
being crowded with small yolk platelets, while only a few large yolk granules are
found in that region. Cytoplasmic islands have been observed mainly in the
subperipheral zone and at the boundary between the subperipheral zone and
the internal mass, very obviously separated from the peripheral layer. The
16
]EEM
22
242
R. CZOLOWSKA
islands are easily distinguishable due to their heavy staining, large size (largest
reaching 16/0 ar) d abundance.
In eggs of the second group taken from the uterus as well as in some unfertilized eggs the pattern of yolk distribution is obliterated (Fig. 8B). The clear
segregation of fine and coarse yolk platelets is not observed; the large platelets
are distributed over the whole section and approach the peripheral layer very
closely (Fig. 5D). The cytoplasmic islands occupy a less fixed position. Generally
diminished in size and number in comparison with the first group, they are
dispersed not only in the subperipheral zone and even into the internal mass but
also often in the peripheral zone, where they are barely recognizable among the
cortical vacuoles.
In both groups the cytoplasmic islands are found along the curvature of the
vegetal cap of the egg varying from 40 to 216 /«• in width. The cytoplasmic
islands seem to be associated with small yolk platelets (Fig. 5 D) and have a fine
granular appearance.
After staining with methyl green-pyronine the cytoplasm of the vegetative
region is barely visible, being dispersed among the yolk platelets. Contrasting
with this background, RNA-rich islands have been found, corresponding in size
and appearance to the cytoplasmic islands described above.
Fertilized eggs at the time of first furrow formation
The same three zones can be easily distinguished in serial tangential sections
of the vegetative pole region. Cortical vacuoles have disappeared from the peripheral zone. The peripheral and subperipheral zones are characterized by the
presence of irregular cytoplasmic islands which differ in size, number and distribution from egg to egg. These islands are found only in a region directly surrounding the vegetative pole covering an area varying from 96 to 174/t in width. By
comparison with previous stages it seems that the cytoplasmic islands which are
originally dispersed over a rather extensive circular area undergo some process
of concentration as soon as the first furrow approaches the vegetative pole.
Eggs with two separated blastomeres show compact islands of material
localized in the neighbourhood of the cleavage furrow (Fig. 5E). Islands are
often grouped lying in a row, like a string of beads, parallel to the egg membrane.
Their size varies from 1 to 22 jn across.
Each cytoplasmic area is associated with groups of small yolk platelets
(Fig. 5E) and contains fine oblong granules.
The highly dispersed cytoplasm in the vegetative half of the egg stains weakly
with methyl green-pyronine. However, islands extremely rich in RNA are
always present, distributed within a definite region. The appearance and size
of these islands seems to be identical with those of the blue cytoplasmic islands
after Azan staining.
Germinal cytoplasm in Xenopus
243
DISCUSSION
Unfertilized and fertilized eggs of Amphibia both show an unequal distribution of yolk concentrated predominantly in the vegetative hemisphere. Consequently there is an apparent decrease in cytoplasm along the animal-vegetative
axis. In the most vegetative region of the egg a cytoplasmic network can barely
be seen. In the previous section, this unequal distribution has been called an
animal-vegetative gradient. Here the term 'gradient' only indicates the pronounced differences in cytoplasm and yolk content along the primary axis, and
does not indicate that a smooth, continuous decline or increase of these components occurs in animal-vegetative direction.
Cytoplasmic islands, apparently different from the dispersed cytoplasm, have
been observed near the vegetative pole of the fertilized eggs of Xenopus laevis
(Nieuwkoop, 1956; Nieuwkoop & Faber, 1956; Blackler, 1958) and identified
as the 'germinal cytoplasm'. These observations have been confirmed in the
present studies. 'Germinal cytoplasm' was found close to the cell membrane in
the most vegetative region of Xenopus eggs fixed at the time of formation of the
first furrow. The islands are easily detectable due to their strong staining with
aniline blue and characteristic internal granules, corresponding in size to mitochondria. The presence of a crowded population of mitochondria as a constant
component of the 'germinal cytoplasm' has been demonstrated previously by
specific staining with Altman's acid fuchsin (Bounoure, 1934; Blackler, 1958;
Berardino, 1961).
The exact time of the initial appearance of the 'germinal cytoplasm' and the
mechanism of its formation has still remained obscure. According to the available data in the literature the 'germinal cytoplasm' appears in the egg shortly
after fertilization; its appearance seems to be correlated with the activation of
the egg rather than with the presence of sperm (Blackler, 1958). Blackler could
not detect the 'germinal cytoplasm' in unfertilized eggs and oocytes of R.
temporaria. Although he does not make any definative statement he mentions
that '"germ plasm" was seen in the process of formation in the vegetal cortical
region', thus implying a cortical origin. However, Bounoure (1964) holds the
opinion that the 'germinal cytoplasm' ... 'prend evidemment naissance au cours
de l'ovogenese, dans cette phase obscure du grand accroissement, ou se fixent
les structures et les potentialites de l'ovule'.
With Bounoure's idea in mind, unfertilized eggs of X. laevis as well as
ovulated oocytes were investigated. Cytoplasmic islands in the most vegetative
region were found in a high percentage of specimens in every group. The following criteria have been selected for their identification and comparison: (1) strong
stainability with aniline blue, (2) localization restricted to a limited sector of the
egg around the vegetative pole, (3) the presence of an internal granulation
resembling mitochondria, and (4) rich in RNA.
The distribution of the islands, as well as their number and size, varies from
lfi-2
244
R. CZOLOWSKA
sample to sample and from female to female; however, they are usually situated
in the subperipheral zone which is laden with small yolk platelets and rich in
pigment granules.
Bladder (1958) has reported the presence of RNA in the 'germinal cytoplasm'
of fertilized, uncleaved eggs of R. temporaria. In the course of the present studies
the same cytochemical test was used for X. laevis eggs. Eggs studied when the
first cleavage furrow is formed revealed the presence of RNA within the
'germinal cytoplasm'. In the present studies unfertilized eggs and ovulated
oocytes likewise showed a positive reaction for RNA within the cytoplasmic
islands distributed around the vegetative pole. These isolated accumulations
of RNA are distinguishable from the general animal-vegetative 'gradient' of
RNA, originally described by Brachet (1940).
Although conclusions drawn from static observations have serious limitations,
it seems reasonable to suppose that the islands observed in groups of eggs taken
sequentially do represent the same material. This material is segregated in a
definite region of the cell in the ovulated oocyte and stored there during its
further development. In its appearance and composition the material resembles
closely the cytoplasmic islands described by Bounoure. It is therefore suggested
that we are dealing throughout with the 'germinal cytoplasm' or, at least, with
its precursor.
On the basis of our present knowledge of the synthesis of nucleic acids all
RNA found in the cell can be considered as a product of the activity of the
nucleus. Therefore, the RNA component of the 'germinal cytoplasm' is thought
to be exclusively of nuclear origin. On the other hand, an extranuclear origin
of the second component of the 'germinal cytoplasm', the mitochondria, seems
plausible.
The characteristic appearance of the 'germinal cytoplasm' could not be recognized earlier than in the ovulated oocyte. This may mean that either (1) the time of
its actual formation corresponds to the ovulation period, or (2) that the time of its
characteristic manifestation coincides with ovulation, the 'germinal cytoplasm',
however, existing already in some other form in the non-ovulated oocyte.
1. Is the 'germinal cytoplasm' formed during maturation?
Ovulation itself is only one of numerous phenomena occurring during maturation of the oocyte. According to Dettlaff, Nikitina & Stroeva (1964) and Briggs
& Cassens (1966) the significance of maturation for the further fate of the egg,
even for processes as remote as cleavage and post-gastrulation, becomes more
and more evident.
It is known that by the end of oogenesis a pronounced inhibition of the
synthesis of nucleic acids and proteins occurs (Monroy, 1967). During maturation the general pattern of synthesis changes.
According to Dettlaff (1966) the initiation of maturation by hormonal
stimulus in R. temporaria acts through the induction of the synthesis of specific
Germinal cytoplasm in Xenopus
245
m-RNA; actinomycin, when applied in the hormone-dependent period, inhibits
maturation. On the contrary, Brachet (1967a) reports that all stages of maturation of the X. laevis oocyte can be inhibited by actinomycin. Protein synthesis,
tested by using pyromycin, seems to be involved in all steps of the maturation
process (Dettlaff, 1966; Brachet, 1967a).
Biochemical and autoradiographical data indicate that small amounts of
heterogeneous, rapidly labelled RNA (probably m-RNA) is indeed synthesized
at the time of maturation (Ficq, 1964; Brown, 1966; Brown & Littna, 1964a,
1966 a). Monroy (1967) reports detectable synthesis of s-RNA and Smith,
Ecker & Subtelny (1966) active synthesis of proteins during maturation.
Brachet (1965, 19676, c) describes certain very interesting phenomena correlated with maturation. Hormone treatment of the isolated oocyte for 3-4 h
leads to changes in the germinal vesicle. The nuclear membrane on the vegetative
side shows numerous invaginations, extremely rich in RNA. The nuclear sap
becomes very basophilic, corresponding to an increase in ribosome content
(unpublished observations of van Gansen, cited by Brachet, 1967 c). The fate
of this particular population of ribosomes is not known, the possibility of their
eventual occurrence in the 'germinal cytoplasm' remaining open.
Unfortunately the ultrastructure of the 'germinal cytoplasm' in Anura has
not yet been intensively studied. Balinsky (1966) has presented the single electron
micrograph demonstrating the vegetative region of the fertilized egg of Phrynobatrachus natalensis. Besides large groups of mitochondria occupying this
region he found 'peculiar rounded bodies about 018 [i in diameter. These bodies
are electron-dense and appear to consist of aggregations of particles which are
smaller than the ribosomes seen in the same area'. According to his suggestion
'these [structures] may represent the basophilic areas of cytoplasm...known as
"germinal cytoplasm"'.
J. Brachet (personal communication) holds the opinion that rapidly labelled
heterogenous RNA may have something to do with the formation of
the 'germinal cytoplasm'. At present this hypothesis is being studied in his
laboratory.
Only ribosomal RNA can be detected with cytochemical methods. In the
light of the present results the participation of r-RNA in the 'germinal cytoplasm' is therefore strongly indicated.
Unfortunately the cytological observations do not provide convincing evidence for the nuclear origin of the RNA component of the 'germinal cytoplasm'.
The author has, indeed, seen sections which suggest a possible transport of
animal material towards the vegetative pole. At the time of ovulation the mass
of vegetative yolk becomes very loose and streams of cytoplasm seem to become
intermingled with yolk platelets. This cytoplasm is always seen as a fibrous,
dispersed network. Material resembling the compact islands of the 'germinal
cytoplasm' has, however, never been observed within the mass of yolk platelets
filling the vegetative hemisphere.
246
R. CZOLOWSKA
There is some doubt how to interpret observations on the oocytes ovulated
in vitro.
Ovulation in vitro occurs under the influence of a high dose of gonadotrophic
hormone. This rather brutal treatment evokes phenomena which develop more
intensively and more rapidly than in the natural ovulation process (Dettlaff,
1966). It remains open how far oocytes ovulated in vitro are a model system
illustrating processes which are normally known to occur in a masked way.
Observations on the oocytes ovulated in vitro reveal a few interesting details
with respect to the 'germinal cytoplasm'. The number of islands as well as the
region of their distribution are significantly greater than those of oocytes
ovulated in vivo. The islands are extremely rich in RNA. The time factor seems
to play an important role in their manifestation and distribution. A high number
of inclusions always remain in close contact with the outermost cytoplasmic
layer of the peripheral zone.
Some sections suggest a possible displacement of the islands within the
vegetative area. The layer containing the vegetative pigment and the small yolk
platelets increases in thickness in the course of maturation. This phenomenon
as well as a general loosening of the yolk could be due to rapid hydration of the
ovulated oocyte (Tchou-Su & Yen Pai-Hu, 1950), resulting from a change in
permeability of the cell membrane (Morrill, 1965; Morrill, Rosenthal &
Watson, 1966). In addition a very sharp increase in the contractility of the
egg surface occurs at the time of the breakdown of the germinal vesicle (Dettlaff,
1966).
Although we are dealing with static pictures the evidence presented above
suggests that in oocytes ovulated in vitro some RNA-rich material, closely
resembling the 'germinal cytoplasm', is localized initially in the peripheral zone
at the vegetative side. This material becomes more clearly visible in the course
of maturation and migrates from the periphery towards deeper layers of the
oocyte.
2. Is the 'germinal cytoplasm' formed in the growing oocyte?
The period of growth of the amphibian oocyte is marked by numerous
morphological phenomena (Wittek, 1952; Wartenberg & Schmidt, 1961;
Balinsky & Devis, 1963; Kemp & Istock, 1967).
A very high rate of synthesis of nucleic acids and proteins occurs in the oocyte
during the growth period. Most of the RNA synthesized at this period consists
of ribosomal RNA which seems to be gradually accumulated during oogenesis
(Brown, 1964, 1966; Brown & Gurdon, 1964; Brown & Littna, 19646; Davidson, Allfrey & Mirsky, 1964; Ficq, 1964). The synthesis of s-RNA proceeds at a
lower level (Brown, 1964; Brown & Littna, 19646, 19666; Ficq, 1964; Monroy,
1967). There is strong evidence that the synthesis and storage of small quantities
of m-RNA also occurs during the growth period (Ficq, 1964; Davidson, Crippa,
Kramer & Mirsky, 1966; Crippa, Davidson & Mirsky, 1967).
Germinal cytoplasm in Xenopus
247
In view of these findings the possibility cannot be excluded that particular
RNA components of the 'germinal cytoplasm' may already be formed in the
period preceding maturation.
Large oocytes isolated from the ovary demonstrate the presence of RNA in
cytoplasmic patches at the vegetative pole region. It is probable that this material
represents a dispersed state of the 'germinal cytoplasm'. In the course of maturation the RNA-containing material would undergo a process of condensation.
It may be concluded that these cytological observations suggest a multiple
origin of the 'germinal cytoplasm'. On the one hand they emphasize the significance of the period of oogenesis in which the actual formation of the 'germinal
cytoplasm' seems to take place. The large ovarian oocytes, mainly, show the
presence of condensations of RNA-containing material before the onset of
maturation. Since all RNA can be considered as a nuclear product, this RNA
component is evidently formed during oogenesis. The constant association of
the 'germinal cytoplasm' with mitochondria may point towards a relationship
with the Balbiani body, which is thought to give rise to the mitochondrial
population of the oocyte (Wittek, 1952; Balinsky & Devis, 1963). It seems
possible therefore that the structures participating in the formation of the
'germinal cytoplasm' have their origin partially in early as well as in later phases
of oogenesis. It also seems likely from these studies that an 'informative' component is added after the breakdown of the germinal vesicle, leading to the
ultimate differentiation of the 'germinal cytoplasm'. The actual time of synthesis of this RNA component may precede considerably its movement towards
the vegetative pole of the egg.
Without elaborating the theoretical implications the author wishes to point
out the possible significance of these observations for the problem of nuclear
versus cytoplasmic inheritance, as this will in part depend upon the character of
the nuclear and cytoplasmic components of the 'germinal cytoplasm' and their
time and mode of formation.
SUMMARY
1. Fertilized eggs, unfertilized eggs, oocytes ovulated in vivo and in vitro as
well as ovarian oocytes were investigated in order to elucidate the origin of the
'germinal cytoplasm'. For its cytological identification meridional and tangential sections were stained by the Azan method and the methyl green-pyronine
of Unna.
2. A high percentage of specimens in all groups of eggs and ovulated oocytes
sequentially studied has shown the presence of cytoplasmic islands in the most
vegetative region of the cell. The islands are located in the peripheral and subperipheral zones of a limited sector of the vegetative pole region. They are laden
with small yolk platelets and rich in pigment. The presence of internal granulation resembling mitochondria seems to be one of the characteristic features of
the islands studied.
248
R. CZOLOWSKA
3. In large ovarian oocytes the cytoplasmic patches are scattered in the
vegetative pole region. These patches are apparently smaller, more dispersed
and less numerous than the corresponding cytoplasmic islands in ovulated
oocytes.
4. It has been shown cytochemically that the cytoplasmic islands in all the
groups studied contain RNA. These isolated accumulations of RNA at the
vegetative pole region are additional to the general animal-vegetative 'gradient'
of distribution of RNA in the oocyte and in the egg.
5. On the basis of the present results it is suggested that the cytoplasmic
patches localized at the vegetative pole region of ovarian oocytes represent the
'germinal cytoplasm', or its precursor, in the unfertilized and fertilized egg.
The nuclear and extranuclear origin of the various components of the 'germinal
cytoplasm' is discussed as well as the possible time of their formation.
RESUME
Observations sur Vorigine du 'cytoplasme germinal* chez
Xenopus laevis
1. Pour elucider l'origine du cytoplasme germinal, on a examine des oeufs
fecondes, des oeufs vierges, des oocytes ovules in vivo et in vitro, ainsi que des
oocytes ovariens. Pour le mettre en evidence cytologiquement, des coupes
tangentielles et meridiennes ont ete colorees a l'Azan et au vert de methylepyronine d'Unna.
2. Un fort pourcentage d'echantillons dans les groupes d'ceufs et d'oocytes
ovules, etudies en coupes seriees, ont montre la presence d'ilots de cytoplasme
dans la region la plus vegetative de la cellule. Ces ilots sont localises dans les
zones peripheriques et subperipheriques d'un secteur limite de la region du
pole vegetatif. Us sont charges de petites plaquettes vitellines et riches en pigment. La presence de granulations internes ressemblant a des mitochondries
semble etre une des caracteristiques des ilots etudies.
3. Dans les gros oocytes ovariens, les taches cytoplasmiques sont dispersees
dans la region du pole vegetatif. Ces taches sont apparemment plus petites, plus
dispersees et moins nombreuses que les ilots cytoplasmiques correspondant
des oocytes ovules.
4. On a montre cytochimiquement que les ilots cytoplasmiques dans tous les
groupes etudies contiennent du RNA. Ces accumulations isolees de RNA dans
la region du pole vegetatif se situent en dehors du 'gradient' general, animalvegetatif, de repartition du RNA dans l'oocyte et l'ceuf.
5. Sur la base des resultats obtenus, on suggere que les taches cytoplasmiques
localisees dans la region du pole vegetatif des oocytes ovariens representent le
'cytoplasme germinal' (ou son precurseur) de Foeuf non feconde et feconde.
L'origine nucleaire et extranucleaire des divers composants du 'cytoplasme
germinal' est discutee, ainsi que le moment possible de leur formation.
Germinal cytoplasm in Xenopus
249
This project was suggested by Professor P. D. Nieuwkoop, Hubrecht Laboratory, to
whom I would like to express my deep gratitude for his lively interest, stimulating advice, and
encouragement during the course of this work.
I am greatly indebted to Dr G. A. Ubbels for her friendly help in the histological procedure
and would like to thank Dr E. C. Boterenbrood for her help in obtaining the material.
1 wish to thank Dr K. A. Lawson for her criticisms and suggestions made during the
correction of the English manuscript.
The author is grateful to Mrs N. Pulle, Central Embryological Library, for her help in
supplying literature.
REFERENCES
AUBRY, R. (1953). Analyse des certaines conditions experimentales favorisant la sterilization
ab ovo de la grenouille rousse. C. r. Seanc. Soc. Bioi, Paris 147, 893-4.
BALINSKY, B. I. (1966). Changes in theultrastructure of amphibian eggs following fertilization.
Acta Embryol. Morph. exp. 9, 132-54.
BALINSKY, B. 1. & DEVIS, R. J. (1963). Origin and differentiation of cytoplasmic structures in
the oocytes of Xenopus laevis. Acta Embryol. Morph. exp. 6, 55-108.
BERARDINO, M. A. DI (1961). Investigations of the germ-plasm in relation to nuclear transplantation. /. Embryol. exp. Morph. 9, 507-13.
BLACKLER, A. W. (1958). Contribution to the study of germ-cells in the Anura. /. Embryol.
exp. Morph. 6, 491-503.
BLACKLER, A. W. (1960). Transfer of germ-cells in Xenopus laevis. Nature, Lond. 185, 859-60.
BLACKLER, A. W. (1961). The transfer of primordial germ-cells in Xenopus laevis. In Germ
Cells and Earliest Stages of Development, pp. 25-8. Symp. Inst. Int. Embryol. and Fond.
Palanza (1960). Milano.
BLACKLER, A. W. (1962 a). Transfer of primordial germ-cells between two sub-species of
Xenopus laevis. Revue suisse Zool. 69, 286-7.
BLACKLER, A. W. (19626). Transfer of primordial germ-cells between two sub-species of
Xenopus laevis. J. Embryol. exp. Morph. 10, 641-51.
BLACKLER, A. W. (1965 a). Germ-cell transfer and sex ratio in Xenopus laevis. J. Embryol. exp.
Morph. 13, 51-61.
BLACKLER, A. W. (19656). The continuity of the germ line in amphibians and mammals.
Annee biol. 4, 627-35.
BLACKLER, A. W. & FISCHBERG, M. (1961). Transfer of primordial germ-cells in Xenopus
laevis. J. Embryol. exp. Morph. 9, 634-41.
BOUNOURE, L. (1927). Le chondriome des gonocytes primaires chez Rana temporaria et la
recherche des elements genitaux aux jeunes stades du developpement. C. r. hebd. Seanc.
Acad. Sci., Paris 185, 1304-5.
BOUNOURE, L. (1934). Recherches sur la lignee germinale chez la Grenouille rousse aux
premiers stades du developpement. Annls Sci. nat. lOe ser. 17, 67-248.
BOUNOURE, L. (1935 a). Sur la possibilite de realiser une castration dans l'oeuf de la Grenouille
rousse; resultats anatomiques. C. r. Seanc. Soc. Biol., Paris 120, 1316.
BOUNOURE, L. (19356). Une preuve experimentale du role du determinant germinal chez la
Grenouille rousse. C. r. hebd. Seanc. Acad. Sci., Paris 201, 1223.
BOUNOURE, L. (1937 a). Le determinant germinal est-il bien en cause dans l'atrophie des
gonades consecutive a Faction des rayons ultraviolets sur le pole inferieur de l'oeuf de
Grenouille? C. r. Seanc. Soc. Biol., Paris 125, 895.
BOUNOURE, L. (19376). Les suites de l'irradiation du determinant germinal, chez la Grenouille
rousse, par les rayons ultra-violets; resultats histologiques. C.r. Seanc. Soc. Biol., Paris 125,
898.
BOUNOURE, L. (1937 C). Le sort de la lignee germinale chez la Grenouille rousse apres Faction des
rayons ultraviolets sur le pole inferieur de l'oeuf. C. r. hebd. Seanc. Acad. Sci., Para 204,1837.
1
BOUNOURE, L. (1937 a ). La constitution des glandes genitales chez la Grenouille rousse apres
destruction etendue de la lignee germinale par Faction des rayons ultraviolets sur l'oeuf.
C. r. hebd. Seanc. Acad. Sci., Paris 204, 1957.
250
BOUNOURE, L. (1939). VOrigine
R. CZOLOWSKA
des cellules reproductrices et le probleme de la lignee germinale.
Paris: Gauthier-Villars.
BOUNOURE, L. (1950). Sur le developpement sexuel des glandes genitales de la Grenouille en
l'absence de gonocytes. Archs Anat. microsc. Morph. exp. 39, 247-56.
BOUNOURE, L. (1964). La lignee germinale chez les batraciens anoures. Jn VOrigine de la
lignee germinale (ed. Et. Wolff), Sem. (1962), pp. 207-34. Paris: Hermann.
BOUNOURE, L., AUBRY, R. & HUCK, M.-L. (1954). Nouvelles recherches experimentales sur
les origines de la lignee reproductrice chez la Grenouille rousse. /. Embryol. exp. Morph. 2,
245-63.
BRACHET, J. (1940). Etude histochimique des proteins au cours du developpement embryonnaire des Poissons, des Amphibiens et des Oiseaux. Archs Bio/., Liege 51, 167-202.
BRACHET, J. (1965). Emission of Feulgen-positive particles during the in vitro maturation of
toad ovocytes. Nature, Lond. 208, 596-7.
BRACHET, J. (1967 a). Effects of actinomycin, puromycin and cycloheximide upon the
maturation of Amphibian ovocytes. Expl Cell Res. 48, 233-6.
BRACHET, J. (19676). Protein synthesis in the absence of the nucleus. Nature, Lond. 213,
650-5.
BRACHET, J. (1967 c). Behaviour of nucleic acids during early development. Tn Comprehensive
Biochemistry. Vol. 28. Morphogenesis, Differentiation and Development (ed. M. Florkin and
E. H. Stotz) pp. 23-54. Amsterdam, London, New York: Elsevier Publ. Comp.
BRIGGS, R. & CASSENS, G. (1966). Accumulation in the oocyte nucleus of a gene product
essential for embryonic development beyond gastrulation. Proc. natn. Acad. Sci. U.S.A. 55,
1103-9.
BROWN, D. D. (1964). RNA synthesis during amphibian development. /. exp. Zool. 157,
101-13.
BROWN, D. D. (1966). The nucleolus and synthesis of ribosomal RNA during oogenesis and
embryogenesis of Xenopus laevis. Natn. Cancer Inst. Monogr. 23, 297.
BROWN, D. D. & GURDON, J. B. (1964). Absence of ribosomal RNA in the anucleolate
mutant of Xenopus laevis. Proc. natn. Acad. Sci. U.S.A. 51, 139-46.
BROWN, D. D. & LITTNA, E. (1964a). RNA synthesis during the development of Xenopus
laevis, the South African clawed toad. J. molec. Biol. 8, 669-87.
BROWN, D. D. & LITTNA, E. (19646). Variations in the synthesis of stable RNAs during
oogenesis and development of Xenopus laevis. J. molec. Biol. 8, 688-95.
BROWN, D. D. & LITTNA, E. (1966a). Synthesis and accumulation of DNA-like RNA during
embryogenesis of Xenopus laevis. J. molec. Biol. 20, 81-94.
BROWN, D. D. & LITTNA, E. (19666). Synthesis and accumulation of low molecular weight
RNA during embryogenesis of Xenopus laevis. J. molec. Biol. 20, 95-112.
CRIPPA, M., DAVIDSON, E. H. & MIRSKY, A. E. (1967). Persistence in early amphibian
embryos of informational RNAs from the lampbrush stage of oogenesis. Proc. natn. Acad.
Sci. U.S.A. 57, 885-92.
DAVIDSON, E. H., ALLFREY, V. G. & MIRSKY, A. E. (1964). On the RNA synthesized during
the lampbrush phase of amphibian oogenesis. Proc. natn. Acad. Sci. U.S.A. 52, 501-8.
DAVIDSON, E. H., CRIPPA, M., KRAMER, F. R. & MIRSKY, A. E. (1966). Genomic function
during the lampbrush chromosome stage of amphibian oogenesis. Proc. natn. Acad. Sci.
U.S.A. 56, 856-63.
DETTLAFF, T. A. (1966). Action of actinomycin and puromycin upon frog oocyte maturation. J. Embryol. exp. Morph. 16, 183-95.
DETTLAFF, T. A., NIKITINA, L. A. & STROEVA, O. G. (1964). The role of the germinal vesicle
in oocyte maturation in anurans as revealed by the removal and transplantation of nuclei.
J. Embryol. exp. Morph. 12, 851-73.
FICQ, A. (1964). Effets de Pactinomycine D et de la puromycine sur le metabolisme de
l'oocyte en croissance. Etude autoradiographique. Expl Cell Res. 34, 581-94.
FISCHIAROLO, G. (1960). La formazione di cellule germinali negli Anfibi anuri dopo distruzione di blastomeri vegetativi della blastula. Atti Accad. naz. Lincei Re. ser. 8, 28, 5.19-21.
GANSEN, P. VAN (1966). Ultrastructure comparee du cytoplasme peripherique des oocytes
murs et des ceufs vierges de Xenopus laevis (Batracien anoure). /. Embryol. exp. Morph. 15,
355-64.
Germinal cytoplasm in Xenopus
251
GIPOULOUX, J.-D. (1962 a). Mise en evidence du 'cytoplasms germinal' dans l'oeuf et l'embryon
du Discoglosse: Discoglossuspictus. C. r. hebd. Seanc. Acad. Set'., Paris 254, 2433-5.
GIPOULOUX, J.-D. (19626). L'ablation de l'endoderme provoque la sterilization de la larvae
chez le Discoglosse (Discoglossuspictus). C. r. hebd. Seanc. Acad. ScL, Paris 254, 4081-2.
HOPE, j . , HUMPHRIES, A. A. & BOURNE, G. H. (1963). Ultrastructural studies on developing
oocytes of the salamander Triturus viridescens. I. The relationship between follicle cells
and developing oocytes. / . Ultrastruct. Res. 9, 302-24.
KEMP, N. E. (1953). Synthesis of yolk in oocytes of Rana pipiens after induced ovulation.
/ . Morph. 92, 487-505.
KEMP, N. E. (1956). Electron microscopy of growing oocytes of Rana pipiens. J. biophys.
biochem. Cytol. 2, 281-92.
KEMP, N. E. & ISTOCK, N. L. (1967). Cortical changes in growing oocytes and in fertilized
or pricked eggs of Rana pipiens. J. Cell Biol. 34, 111-22.
LIBRERA, E. (1964). Effects on gonad differentiation of the removal of vegetal plasm in eggs
and embryos of Discoglossus pictus. Acta Embryol. Morph. exp. 7, 217-33.
MORRILL, G. A. (1965). Water and electrolyte changes in amphibian eggs at ovulation.
Expl Cell Res. 40, 664-7.
MORRILL, G. A., ROSENTHAL, J. & WATSON, D. E. (1966). Membrane permeability changes
in amphibian eggs at ovulation. / . Cell Physiol. 67, 375-82.
MONROY, A. (1939). Sulla localizzazione delle cellule genitali primordiali in fasi precoci di
sviluppo. Ricerche sperimentali in Anfibi Anuri. Archo ital. Anat. Embriol. 41, 368-89.
MONROY, A. (1967). Fertilization. In Comprehensive Biochemistry. Vol. 28. Morphogenesis,
Differentiation and Development (ed. M. Florkin and E. H. Stotz), pp. 1-21. Amsterdam,
London, New York: Elsevier Publ. Comp.
NIEUWKOOP, P. D. (1956). Are there direct relationships between the cortical layer of the
fertilized egg and the development of the future axial system in Xenopus laevis embryos?
Pubbl. Staz. zool. Napoli 28, 241-9.
NIEUWKOOP, P. D. & FABER, j . (1956). Normal Table o/Xenopus laevis (Daud.). Amsterdam:
North Holland Publ. Co.
NIEUWKOOP, P. D. & SUMINSKJ, E. H. (1959). Does the so called 'germinal cytoplasm' play
an important role in the development of the primordial germ cells? Archs Anat. microsc.
Morph. exp. 48 bis, 189-98.
PADOA, E. (1963). Le gonadi di girini di Rana esculenta da uova irradiate con ultravioletto.
Monitore Zool. ital. 70-71, 238-49.
PADOA, E. (1964). Qualche precisazione sulla possibilita'di distruggere con l'ultravioletto il
plasma germinale delle uova di Rana esculenta. Boll. Soc. ital. Biol. sper. 40, 272-5.
ROMEIS, B. (1948). Mikroskopische Technik. Miinchen: Leibniz.
SELMAN, G. G. & PAWSEY, G. J. (1965). The utilization of yolk platelets by tissues of Xenopus
embryos studied by a safranin staining method. / . Embryol. exp. Morph. 14, 191-212.
SMITH, L. D. (1966). The role of a 'germinal plasm' in the formation of primordial germ cells
in Rana pipiens. Devi Biol. 14, 330-47.
SMITH, L. D., ECKER, R. E. & SUBTELNY, S. (1966). The initiation of protein synthesis in eggs
of Rana pipiens. Proc. natn. Acad. Sci. U.S.A. 56, 1724-8.
TCHOU-SU & YEN PAI-HU (1950). Relation de l'augmentation d'eau dans l'oeuf et la maturation. Scientia Sin. 1, 165-83 Cited by Dettlaff et ai, (1964).
TOKIN, I. B. & GABAYEVA, N. S. (1963). Electron-microscope analysis of cortical parts of the
oocytes of Rana temporaria. Vestn. Leningr. Univ. 15, 158-60.
UNNA, P. G. (1913). Die Herkunft der Plasmazellen. Virchows Arch. path. Anat. Physiol. 214,
320-39.
WARTENBERG, H. & SCHMIDT, W. (1961). Elektronenmikroskopische Untersuchungen der
strukturellen Veranderungen im Rindenbereich des Amphibieneies im Ovar und nach der
Befruchtung. Z. Zellforsch. 54, 118-46.
WITTEK, M. (1952). La vitellogenese ches les Amphibiens. Archs Biol., Liege 63, 133-98.
{Manuscript received 8 October 1968)