/ . Embryol. exp. Morph. Vol. 39, pp. 221-233, 1977
Printed in Great Britain
221
Partial characterization of ; primordial
germ cell-forming activity' localized in vegetal pole
cytoplasm in anuran eggs
BY MASAMI WAKAHARA 1
From the Zoological Institute, Hokkaido University, Japan
SUMMARY
Larvae of Rana chensinensis developed from fertilized eggs which had been subjected to
ultraviolet (u.v.) irradiation on their vegetal hemisphere at a dose of 20000 ergs/mm2 within
60 min of fertilization contained no primordial germ cells (PGCs) when examined histologically at the stage when the operculum was complete (8 days after fertilization at 18 °C,
stage 25 according to Shumway, 1940). The morphogenetic ability of vegetal pole cytoplasm
from non-irradiated eggs to establish the PGCs was tested by injecting some fractions of this
cytoplasm into the vegetal hemisphere of u.v.-irradiated eggs. Crude homogenate of the
vegetal pole cytoplasm without large yolk platelets was able to restore the PGCs when
injected into u.v.-irradiated eggs, but a similar fraction from animal half cytoplasm had no
ability to form PGCs. The 'PGC-forming activity' demonstrated in the crude homogenate
of the vegetal pole cytoplasm was not abolished by dialysis, lyophilization and heating to
90 °C for 10 min. When the homogenate was fractionated by differential centrifugation in
0-25 M sucrose, the 'PGC-forming activity' was recovered mainly in the precipitate of
15000g for 30 min. The precipitate of 7000 g for 10 min had also a little 'activity'. The
possibility was discussed that the 'PGC-forming activity' demonstrated in the vegetal pole
cytoplasm was associated with the germinal granules in the germ plasm rather than the
mitochondria.
INTRODUCTION
Since the classical work of Bounoure (1934) on germ plasm in Rana temporaria
eggs, the germ plasm has been described repeatedly in various anuran eggs (for
review, see Beams & Kessel, 1974; Eddy, 1975). These descriptive studies led
to the conclusion that the germ plasm, localized in the vegetal pole cytoplasm,
becomes restricted to a few cells embedded in endoderm during cleavage stages
(Nieuwkoop & Faber, 1956; Blackler, 1958; Di Berardino, 1961; Gipouloux,
1962; Czolowska, 1969); and hence the 'presumptive primordial germ cells'
(Kerr & Dixon, 1974) or 'germinal plasm bearing cells' (Ikenishi & Kotani,
1975) migrate to the developing germinal ridges where they differentiate into
the primordial germ cells (PGCs) (Blacker, 1958; Whitington & Dixon, 1975;
Kamimura, Ikenishi, Kotani & Matsuno, 1976; Wylie & Heasman, 1976). The
concept that all the functional gametes develop from the presumptive PGCs
has been proved by means of the 'germ cell transfer' technique (Blackler &
Fishberg, 1961; Blackler, 1962, 1965).
1
Author's address: Zoological Institute, Faculty of Science, Hokkaido University, Sapporo
060, Japan.
15
E MB 39
222
M. WAKAHARA
Smith (1966) has shown that ultraviolet (u.v.) irradiation of the vegetal
hemisphere of Rana pipiens eggs during early cleavage stages causes a complete
absence of PGCs when examined later at tadpole stages, while some PGCs are
reestablished after vegetal pole cytoplasm from non-irradiated eggs is injected
into the vegetal hemisphere of the irradiated eggs. From these results he concluded that the factor indispensable for forming the PGCs, the 'germinal
determinant', is restricted to the vegetal pole cytoplasm. Further, from the
results of the spectrum analysis of the u.v. employed for eliminating the
PGCs, he proposed that the chemical nature of this material is nucleic acid,
such as RNA. Buehr & Blackler (1970) have strongly suggested, as a result of
experiments involving substantial removal of the germ plasm in Xenopus eggs,
that the germ plasm is all important for the formation of PGCs and that it
really contains the 'germinal determinant'. This concept is supported by
recent work which shows that the amount of the germ plasm correlates with
the resulting number of the PGCs (Tanabe & Kotani, 1974; Whitington &
Dixon, 1975).
However, the direct evidence to prove the germ plasm actually specifies the
PGCs or germ line cells is very poor. In this context, it seems necessary to
investigate certain morphogenetic activities of the germ plasm in order to
clarify its biological role. This work has been directed initially towards determining whether specific fractions of the vegetal pole cytoplasm possess an
activity to establish the PGCs. The present paper reports changes of the number
of PGCs in experimental tadpoles, which were initially subjected to u.v. irradiation and then received microinjections of a variety of fractions from unirradiated
vegetal pole cytoplasm.
MATERIALS AND METHODS
Grass frogs, Rana chensinensis, which were collected in the vicinity of Sapporo
during the breeding season, were used as material. Fertilized eggs, which were
prepared by artificial insemination (Katagiri, 1961), were immersed in 1 %
cysteine-HCl solution (adjusted to pH 7-6 by adding Tris) till the jelly layers
were completely dissolved. The dejellied eggs were transferred to Steinberg's
solution (Steinberg, 1957) and allowed to develop within the vitelline membrane
until they were used as recipients of microinjection, or as a source of microinjection materials. The overall plan of my experiments is illustrated in Fig. 1.
U.v. irradiation
The dejellied eggs were placed on quartz slides, as free from water as possible,
with the animal hemisphere upwards. Then, the vegetal halves of the eggs were
irradiated with u.v. at a wavelength of 237-5 nm, given upwardly from a germicidal lamp placed under the slides. The dosage of u.v. given to the eggs was
controlled by changing the duration of irradiation (in my system, about
200 ergs/mm2/sec was obtained at the level of the quartz slides).
c
Primordial germ cell-forming activity' in anuran eggs
223
Dejelly
Preparation of -^^"^
microinjection materials
Microinjection
experiments
Animal part
cytoplasm "^~JB
M
Vegetal pole
cytoplasm
t M
M
U.v. irradiation
Homogenize
Centrifuge
\
Crude homogenates
Heating
Dialysis
Lyophilization
Microinjection
t"
Cultivation of
embryos
if
Differential
centrifugations
/
700 g
7000 g
\ 15 000 ^
Histological examination for
counting the PGCs
Fig. 1. Schematic representation of whole plan of microinjection experiments.
Fertilized, dejellied eggs were used in two different ways, as a source of microinjection materials, or as recipients of microinjection after u.v. irradiation. For
further explanation, see text.
Microinjection
U.v. irradiated eggs were set in a depression hole, of the same diameter as
the eggs, bored in a soft paraffin bed, with the vegetal pole upwards. Under the
stereoscopic dissecting microscope, about 100 nl of microinjection materials
were injected into the subcortical cytoplasm near the vegetal pole of the recipient eggs with a glass micropipette. Injected eggs were allowed to develop at
18 °C in Steinberg's solution during the early stages to blastula and in 1/10
Steinberg's solution thereafter. All glassware was autoclaved and antibiotics
were added to the culture media (100 i.u. penicillin G and 0-1 mg streptomycin
sulphate/ml).
Microinjection materials
When dejellied eggs approached the 2-cell stage (about 150 min after fertilization), they were frozen by immersion in 95 % ethyl alcohol chilled to - 30 °C.
The frozen eggs were divided into a vegetal pole cytoplasmic part (which included
a quarter of the whole egg) and an animal part (which was the remnant three
quarters of the egg) with a razor blade at - 10 °C. The vegetal pole and animal
15-2
224
M. WAKAHARA
part cytoplasm from about 1000 eggs was collected separately, and suspended
in 5 volumes of ice-chilled Steinberg's solution. After homogenization in a
Potter homogenizer, the homogenates were centrifuged gently (80 g for 10 min),
and then the supernatants were employed as the first microinjection material
(step-1 sample; crude homogenates from the vegetal pole cytoplasm and animal
part cytoplasm). The step-1 sample from the vegetal pole cytoplasm was
dialyzed against 1/10 Steinberg's solution for 12 h at 4 °C and then lyophilized
(step-2 sample) or was heated at 60 °C for 10 min (step-3 sample). Further, a
heated (90 °C for 10 min)-dialyzed-lyophilized sample was also prepared
(step-4 sample).
Centrifugations
Frozen vegetal pole cytoplasm was suspended in cold 0-25 M sucrose with
5 mM Tris-HCl buffer (pH 7-4), homogenized and fractionated with differential
centrifugation. After two gentle centrifugations the supernatant was added to
the same volume of 0-35 M sucrose and centrigufated at 700 g for 10 min (the
precipitate was step-5 sample); the supernatant was centrifuged at 7000g
for 10min (the precipitate, step-6 sample); and the supernatant was again
centrifuged at 15000g for 30 min (the precipitate, step-7 sample).
Histology
When injected embryos reached the stage at which the operculum was
complete (8 days after the fertilization, stage 25 according to Shumway, 1940),
they were fixed with Bouin's fluid. Tissues except for head and tail regions were
sectioned transversely in paraffin at 8 /im thickness, and stained with Delafield's
hematoxylin and eosin. Direct PGC counting was performed by tracing all the
PGCs arrived at the developing germinal ridges through serial sections.
RESULTS
U.v. irradiation with various dosages at different developmental stages
Dejellied, fertilized eggs from one batch were subjected to u.v. irradiation of
various dosages (0, 8000, 12000, 16000 and 20000 ergs/mm2) at 60, 120, 180
and 240 min after the fertilization. The stages of the eggs obtained at these
times were just after the second polar body emission, prior to the first cleavage,
2-cell and 4-cell stages, respectively. Almost all eggs, even ones subjected to
higher dose of u.v. irradiation, divided normally and were able to reach stage
25 by 8 days after fertilization, when the number of PGCs was counted.
Fig. 2 shows the average number of PGCs in the developing germinal ridges
of the resulting tadoples of each experimental group. It is clearly shown that the
number of PGCs decreases with increase of u.v. dosage and that it reaches
substantially zero with the higher dose of u.v., when the irradiation was performed within 60 min of fertilization. However, when the eggs were irradiated
' Primordial germ cell-forming activity' in anuran eggs
225
70
60
50
8
40
a,
<_
o
6 30
20
120 min
10
8000
12000
16000
U.v. dose (ergs/mm2)
60 min
20000
Fig. 2. Average number of primordial germ cells (PGCs) per tadpole. Eggs were
irradiated at 60, 120, 180 and 240 min after fertilization with various doses of u.v.,
and allowed to develop to the stage when the operculum was complete (Shumway's
stage 25). Number of tadpoles examined is given in parentheses. All eggs were
obtained from one batch.
at later stages of development, a lesser decrease of the number of PGCs was
observed even after irradiation with the higher dose of u.v.
Microinjection of cytoplasmic fractions
(1) Crude homogenate
The results are summarized in Tables 1 and 2. Out of 127 eggs used in this
experiment, 79 embryos survived to stage 25, and then 36 of these tadpoles
were examined histologically to determine the numbers of PGCs.
Although I failed to induce complete absence of PGCs in the u.v. treated
tadpoles since the u.v. irradiation in this experiment was performed a little
late, the number of PGCs observed in the u.v.-treated group was significantly
low compared with that in intact control tadpoles. All tadpoles developed from
the eggs which had been irradiated with u.v. and injected with crude homogenate from the vegetal pole cytoplasm (step-1 sample) contained a large
number of PGCs in the developing germinal ridges. However, tadpoles which
had been irradiated and injected with a similar fraction from the animal part
cytoplasm had substantially zero PGCs. Steinberg's solution, also, had no
effect on the number of PGCs when injected into the irradiated eggs. The
results of this experiment show convincingly that the crude homogenate from
20000
20000
20000
0
2
3
4
5
25
25
25
26
27
No. of eggs
injected
25
(100)
25
(100)
22
(88)
25
(100)
(92)
23
(88)
22
(64)
16
(65)
17
(70)
19
Tail bud
(88)
22
(68)
18
(42)
12
(62)
16
(41)
11
Operculum
complete
4
5
1
2
3
Serial no.
of experiments
U.v. + vegetal pole
U.v. + animal half
U.v. + Steinberg's
solution
U.v. control
Intact control
Type of
experiment
8
7
7
8
6
No. of
tadpoles
examined
17
33
42
8
5
1
47
29
0
0
27
48
43
0
18
21
61
27
0
0
21
67
37
0
15
5
66
61
7
0
11
71
28
0
—
No. of PGCs in developing germ ridges
2
—
—
0
—
56-3
131
381
1-8
6-3
Average
Table 2. The number of PGCs in the tadpoles developed from eggs microinjected with crude homogenates from vegetal pole and
animal half cytoplasms
(92)
23
(88)
22
(96)
24
25
(100)
25
(100)
(92)
25
(96)
25
24
(96)
26
Gastrula
(96)
26
(96)
27
Blastula
(100)*
Cleavage
Developmental stage reached
* In parentheses, percentage survival of initial number.
Vegetal
pole
Animal
half
Steinberg's
solution
None
20000
1
None
Materials
injected
U.v. dose
(ergs/mm2)
Serial no. of
experiments
Treatment
Table 1. The development resulting from the microinjections of crude homogenates from vegetal pole
and animal half cytoplasms into u.v. irradiated eggs
>
to
' Primordial germ cell-forming activity' in anuran eggs
227
vegetal pole cytoplasm of the unirradiated, fertilized eggs contains an activity
to restore PGCs when injected into the u.v. irradiated eggs.
(2) D iaiyzed, lyophilized and heated fractions
Tables 3 and 4 show the developmental results from the microinjections of
dialyzed, lyophilized and heated homogenates of the vegetal pole cytoplasm
(step 2, 3 and 4 samples). The results were combined from two series of experiments in which eggs from two different batches were used. Since a number of
experimental embryos died from unknown causes in the course of development,
especially during later embryogenesis after the tail-bud stage, the survival rate
was relatively low (see experiment no. 11 in Table 3). Out of 131 eggs used in
these experiments, 63 embryos survived to stage 25, and 44 of these tadpoles
were examined histologically.
The number of PGCs in the tadpoles injected with dialysed and lyophilized
homogenate from the vegetal pole cytoplasm (step-2 sample) was significantly
larger than the number when Steinberg's solution had been used (Table 4). No
differences were detected between the numbers of PGCs in experiment no. 7
(injected with step-3 sample) and no. 8 (injected with step-4 sample), both of
which showed relatively large number of PGCs restored. The results of these
experiments indicate that an activity to restore PGCs is not inactivated by
dialysis, lyophilization and even after heating.
(3) Cytoplasm fractionated by centrifugation
Tables 5 and 6 show the developmental results from the microinjections of
fractions of the vegetal pole cytoplasm obtained by differential centrifugation
(step-5, -6 and -7 samples). Out of 151 eggs used in this experiment, 74 embryos
survived to stage 25, and 33 of these tadpoles were examined histologically.
The number of PGCs in the tadpoles which had been irradiated with u.v. and
received the step-7 sample (a precipitate of 15000g for 30 min centrifugation)
was as high as in the intact control tadpoles. The tadpoles injected with the
step-6 sample (a precipitate of 7000 g for 10 min) contained more PGCs than
the u.v. control tadpoles. However, the number of PGCs in the tadpoles
injected with step-5 sample (700 g for 10 min) was much smaller than in u.v.
controls.
DISCUSSION
This investigation confirms the early observations in several anuran species
that u.v. irradiation of a vegetal pole of fertilized eggs results in a quantitative
change in the number of PGCs when examined later in tadpole stages (in Rana
temporaria, Bounoure, Aubry & Huck, 1954; in R. esculenta, Padoa, 1963; in
R. pipiens, Smith, 1966; in Xenopus laevis, Tanabe & Kotani, 1974; Ziist &
Dixon, 1975; Ijiri, 1976). Smith (1966) has stated that the most effective stage
for u.v. irradiation for the elimination of PGCs is just prior to the first cleavage
None
20000
20000
20000
0
8
9
10
11
25
24
20
23
13
26
No. of eggs
injected
21
22
(88)
25
(100)
24
24
(100)
24
(100)
(88)
22
(100)
(90)
18
(90)
18
(91)
19
21
(91)
23
(100)
7
(54)
(95)
10
(77)
13
(88)
(100)
23
23
(88)
Gastrula
25
Blastula
(96)f
Cleavage
(76)
19
(100)
24
(65)
13
(65)
15
(46)
6
(73)
19
Tail bud
Developmental stage reached
(32)
8
(83)
20
(45)
9
(45)
10
(31)
4
(42)
11
Operculum
complete
* This table includes the combined results of two series of experiments in which two different batches of eggs were used,
f In parentheses, percentage survival of initial number.
Heated (90 °C)
dialysedlyophilized
Steinberg's
solution
None
20000
7
Dialysedlyophilized
Heated (60 °C)
Materials
injected
20000
U.v. dose
(ergs/mm2)
6
Serial no. of
experiments
Treatment
Table 3. The development resulting from the microinjections of experimentally treated homogenates from vegetal pole
cytoplasm into u.v. irradiated eggs*
X
•
5;
OO
to
U.v. + dialysedlyophilized
U.v. + heated
(60 °C)
U.v. + heated
(90 °C)-dialysedlyophilized
U.v. + Steinberg's
solution
U.v. control
Intact control
Type of
experiment
38
0
31
3
1
101
10
14
36
0
29
57
4
4
7
6
5
11
102
1
38
59
1
6
79
0
20
78
13
K
3
85
6
—
—
6
—
—
—
12
—
—
27
—
—
3
10
—
No. of PGCs in developing germ ridges
6
No. of
tadpoles
examined
100
84-8
3-6
31-2
37-8
15-8
Average
* This table includes the combined results of two series of experiments in which twoi different batches of eggs were used.
10
11
9
8
7
6
Serial no.
of experiments
Table 4. The number of PGCs in the tadpoles developed from eggs microinjected with experimentally treated homogenates
from vegetal pole cytoplasm*
I
S"
I
2.
Si
230
M. WAKAHARA
in Rana pipiens. The results on R. chensinensis, however, show that earlier
irradiation with u.v. is more efficient for eliminating the PGCs. Recently, Ijiri
(1976) has reported in X. laevis that u.v. irradiation is most effective when
given just after fertilization. Because u.v. treatment in my experiments has been
effected after the removal of jelly layers, to facilitate the injection procedure,
we cannot determine more precisely the stages specificity of the u.v. action.
Since the PGCs observed in the developing germinal ridges of tadpoles are
abolished by irradiating the vegetal hemisphere of eggs with an appropriate dose
of u.v., and partial restitution of PGCs is obtained by injecting vegetal pole
cytoplasm (Smith, 1966), it has been suspected that the factor indispensable for
forming the PGCs, the 'germinal determinant', is localized in the vegetal pole
cytoplasm. The results presented in the first microinjection experiment (Table
2) provide direct evidence that the morphogenetic activity for the restoration
of PCGs, limited to the vegetal pole cytoplasm, remains in the crude homogenate from the frozen cytoplasm. Although a direct transfer of subcortical
cytoplasm from the vegetal hemisphere into u.v. irradiated eggs by Smith
(1966) resulted in a partial restitution of the PGCs, both the proportion of
animals with the PGCs and the number of PGCs restored per animal were very
small (see Table 3, in Smith, 1966). In contrast to these findings, my results
from microinjections show that crude homogenate from the vegetal pole
cytoplasm without large yolk platelets has a very high PGC-restoring activity.
This suggests that the yolk platelets have no activity for the restoration of
PGCs and that the 'PGC-forming activity' is more concentrated in the crude
homogenate without yolk platelets than in the whole intact vegetal pole
cytoplasm.
The results of the second microinjection experiment (Table 4) show that the
'PGC-forming activity' in the crude homogenate from the vegetal pole cytoplasm is not abolished even after dialysis, lyophilization and heating. From
these results it is concluded that heat-stable and non-dialysable material(s)
possess this 'activity'. In this respect, Smith (1966) has argued from the results
of the spectrum analysis of the u.v. employed for elimination of the PGCs that
the 'germinal determinant' must be nucleic acid, most likely RNA. The results
are in agreement with Smith's proposal, in view of the heat-stability and the
presumed large molecular size of the factor.
Because the germ plasm, which is believed to contain the 'germinal determinant' (Buehr & Blackler, 1970), is limited to the subcortical region near the
vegetal pole of the eggs (Blackler, 1958; Czolowska, 1969; Buehr & Blackler,
1970), it is reasonable to suspect that the 'PGC-forming activity' recovered in
15 000 g fraction from the vegetal pole cytoplasm (Table 6) should be associated
with certain organelles of the germ plasm. Electron microscopic studies on the
germ plasm have shown it is constituted mainly from large numbers of mitochondria and relatively electron dense small structures called dense bodies
(Mahowald & Hennen, 1971; Williams & Smith, 1971; Czolowska, 1972) or
7000 £*
15000#*
None
None
20000
20000
20000
0
13
14
15
16
28
(90)t
20
(91)
34
(94)
32
(100)
28
(90)
21
(96)
36
(100)
32
(100)
20
(100)
20
(100)
Blastula
Cleavage
* Precipitates of these centrifugations.
t In parentheses, percentage survival of i
20
32
36
22
31
No. of eggs
injected
27
(87)
19
(86)
33
(91)
32
(100)
20
(100)
Gastrula
14
(70)
18
(90)
21
(66)
27
(84)
16
(44)
21
(58)
10
(45)
12
(60)
12
(39)
Operculum
complete
19
ail bud
(61)
Developmental stage reached
Type of
experiment
U.v. + 700 g*
XJ.\. + 1000g*
U.v. + 15 000 g*
U.v. control
Intact control
Serial no.
of experiments
12
13
14
15
16
3
6
40
13
69
1
21
44
0
36
8
24
37
10
48
0
0
55
16
43
8
33
53
1
51
11
10
46
21
42
29
No. of PGCs in developing germ ridges
* Precipitate of these centrifugations.
6
9
6
6
6
No. of
tadpoles
examined
13
5-2
15-4
45-8
101
48-2
Average
Table 6. The number of PGCs in the tadpoles from eggs microinjected with fractionated vegetal pole cytoplasm
100 g*
Materials
injected
20000
U.v. dose
(ergs/mm2)
12
Serial no. of
experiments
Treatment
Table 5. The development resulting from the microinjections of fractionated vegetal pole cytoplasm with a series
of centrifugations into u.v. irradiated eggs
232
M. WAKAHARA
germinal granules (Kalt, 1973; Ikenishi, Kotani & Tanabe, 1974; Ikenishi &
Kotani, 1975). The preliminary electron microscopic observations of the
fractionated cytoplasm show that the precipitate of 700 £ for 10-min centrifugation is exclusively occupied with small yolk platelets, that of 7000 g for
10 min contains a large amount of mitochondria and small accumulations of
electron dense, minute granules scattered among the mitochondria. The
precipitate of 15000g for 30 min which corresponds to the most effective fraction, for the restoration of PGCs, when injected into u.v. irradiated eggs,
contains a large amount of membranous structures of unknown origin, broken
mitochondria and some aggregations of electron-dense, minute granules. The
electron-dense granules observed (more in the 15000g- than in the 7000 g
fractions) are similar to the germinal granules in the germ plasm. Between the
two major components of the germ plasm, the mitochondria and the germinal
granules, the former are unlikely candidates for the source of the 'PGC-forming
activity', because the 7000# fraction contains only a weak 'activity' in spite of
a large accumulation of mitochondria withdrawn in it, whereas the 15000g
fraction, which is almost free from mitochondria, has the highest 'activity'.
Among the cytoplasmic components recovered in the 15000g fraction, only
the aggregation of electron dense granules is a common cytoplasmic structure
observed in the germ plasm. Thus though no direct evidence is available, it is
suspected that the source of the 'PGC-forming activity' is the germinal granules
in the germ plasm. More precise fractionation with centrifugation of the vegetal
pole cytoplasm should make it possible to determine the true organelle(s)
bearing the 'PGC-forming activity'.
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BLACKLER, A. W. (1958). Contribution to the study of germ-cells in the anura. /. Embryol.
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'Primordial germ cell-forming activity'' in anuran eggs
233
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(Received 23 November 1976, revised 10 January 1977)