/ . Embryol. exp. Morph. Vol. 31, I, pp. 89-98, 1974
gO,
Printed in Great Britain
Relationship between the amount
of the ' germinal plasm' and the number of
primordial germ cells in Xenopus laevis
ByKAZUYUKI T A N A B ^ A N D MINORU KOTANI2
From the Department of Biology, Osaka City University, Japan
SUMMARY
Tadpoles of Xenopus laevis completely lacking primordial germ cells were obtained by
irradiating the vegetal hemisphere of early 2-cell eggs with u.v. (wavelength, 253-7 nm; dose,
ca. 6000 ergs/mm2). An increasing number of primordial germ cells were observed as the
stage at irradiation advanced from early 2-cell to early 4-cell stages. Furthermore, early
2-cell eggs irradiated with doses ranging from 750 to 6000 ergs/mm2 grew into tadpoles
carrying a decreasing number of primordial germ cells in accord with the increase of the dose.
On the other hand, tadpoles developed from eggs irradiated immediately after being centrifuged at 150 g for 1 min at early 2-cell stage to displace the 'germinal plasm' deeper into the
cytoplasm, carried a considerable number of primordial germ cells. These facts were interpreted to suggest the presence of u.v.-sensitive germ cell determinant in the 'germinal plasm'.
It was revealed by varying the area of irradiation that the number of primordial germ cells
decreased in direct proportion to the increase of the area irradiated. It was concluded that the
amount of the u.v.-sensitive material(s) contained in the 'germinal plasm' determined the
number of primordial germ cells in tadpoles.
INTRODUCTION
Bounoure (1934) first described in Rana temporaria a specially staining cytoplasm (the so-called 'germinal plasm') located in the peripheral cytoplasm
around the vegetal pole of the fertilized eggs. Since then, several authors have
studied the behaviour of the 'germinal plasm' during the normal course of
embryonic development in histological sections of a wide variety of anuran
eggs using various staining methods (Nieuwkoop & Faber, 1956; Blackler,
1958; DiBerardino, 1961; Gipouloux, 1962).
This plasm becomes incorporated into some of the vegetal blastomeres during
early developmental stages, and appears deep in the entoderm at the gastrula
and neurula stages. These cells migrate later into the genital ridges, where they
eventually differentiate into primordial germ cells (Blackler, 1958).
1
Author's address: Department of Medical Zoology, Osaka City University Medical
School, Asahi-machi, Abeno-ku, Osaka 545, Japan.
2
Author's address: Department of Biology, Faculty of Science, Osaka City University,
Sugimoto-cho, Sumiyoshi-ku, Osaka 558, Japan.
90
K. TANABE AND M. KOTANI
Bounoure, Aubry & Huck (1954), on the other hand, obtained animals
without germ cells or partially sterile animals with a reduced number of germ
cells by irradiating the vegetal hemisphere of fertilized eggs with ultraviolet
light (u.v.). In 1966 Smith irradiated early 2-cell eggs with a larger dose of
u.v. and found that all his experimental tadpoles (Rana pipiens) were completely
lacking in primordial germ cells. He showed, further, that primordial germ cells
were able to be reformed when irradiated eggs were injected with vegetal
subcortical cytoplasm of unirradiated eggs. It was then concluded that some
kind of u.v.-sensitive material(s) indispensable for differentiation of primordial
germ cells was located near the vegetal pole. It was not known, however, whether
this material(s) corresponded to the 'germinal plasm', since the 'germinal
plasm' had not been demonstrated in early cleavage stages of Rana pipiens
(DiBerardino, 1961; see Smith, 1966).
On the other hand, the question whether the 'germinal plasm' really contained
a germ cell determinant was investigated by Buehr & Blackler (1970), who
removed the 'germinal plasm' together with some of the vegetal cytoplasm by
pricking the egg near the vegetal pole. The result, that the proportion of embryos completely lacking 'germinal plasm' at blastula stage nearly equalled
that of tadpoles lacking primordial germ cells, suggested a close relationship
between the presence of the 'germinal plasm' and the formation of primordial
germ cells. It was also suggested that partially sterile tadpoles were likely to
have developed from operated eggs whose 'germinal plasm' was only partially
removed. It is hard to define, however, what really was removed, quantitatively
and qualitatively, through these microsurgical operations.
In the present study, an advantage was taken of the fact that, with u.v., it
should become much easier to inactivate a known proportion of the 'germinal
plasm'. A close relation between the 'germinal plasm' and the u.v.-sensitive
'germinal determinant' was demonstrated by a combination of irradiation and
centrifugation procedures. It was also examined, by varying the area of irradiation, whether a quantitative correlation existed between the amount of the
intact 'germinal plasm' and the number of primordial germ cells formed.
MATERIALS AND METHODS
Fertilized eggs of Xenopus laevis were obtained by injecting human chorionic
gonadotrophic hormone into mature toads (200 units for males, and 300 units
for females). Embryos were staged after Nieuwkoop & Faber (1956).
U.v. irradiation
Eggs at stage 2~ (beginning of the first cleavage), 2 and 3~ (beginning of the
second cleavage) and eggs centrifuged at stage 2~ were subjected to u.v. irradiation. In order to irradiate the vegetal hemisphere, eggs with jelly coats were
placed on quartz slides without excess water, with the animal hemisphere up-
Primordial germ cells in Xenopus
91
Egg with jelly coat
. Quartz slide
• u.v. filter
mini
Germicidal lamp
wavelength 253-7 nm
Aluminum adhesive tape
O Q&r®m.:-:
.
3
4
,
2
,
4
-
Quartz slide
Proportion of vegetal
hemisphere irradiated
Fig. 1. Schematic representation of u.v. irradiation. (A) Method of u.v. irradiation
(side view). (B) Irradiation of proportions of the vegetal hemisphere (upper view).
wards. Then the slides were laid on the band-path filter (Corning Glass Works,
7-54) fixed horizontally just above the light source, Nikko Sekiei Works' SuperLight' LS-D1 (Fig. 1 A). The dose rate at a wavelength of 253-7 nm was about
150ergs/mm2/sec (1 erg = 10-7J) when measured on the surface of the slide.
A total sterility was obtained with a dose of about 6000 ergs/mm2.
In order to examine the relationship between the amount of the intact
(unirradiated) 'germinal plasm' and the number of primordial germ cells,
known proportions of the vegetal hemisphere of stage 2~ eggs were irradiated
by locating eggs at various sites on the edges of aluminium adhesive tape, which
was ascertained beforehand to cut u.v. off completely (Fig. 1B).
Centrifugation
To displace the 'germinal plasm' without disturbing subsequent development, eggs at stage 2~ with jelly coats were placed, animal pole upwards, at the
bottom of a 10 ml centrifuge tube filled with distilled water and centrifuged
at 150 g for 60 sec. Eggs which completed the first cleavage during centrifugation procedures were discarded.
A number of the centrifuged eggs were killed immediately after centrifugation
to see the location of the' germinal plasm'. The rest, with or without subsequent
irradiation, were allowed to develop into feeding tadpoles, where the degree of
formation of primordial germ cells was examined.
92
K. TANABE AND M. KOTANI
Histology
Sterility or fertility of experimental animals was checked as follows. As
aliquots of irradiated and unirradiated batches of eggs derived from the same
mother were allowed to develop into feeding tadpoles (stage 46), those showing
an abnormal appearance were discarded eventually so that only those looking
normal throughout development were fixed with Bouin's fixative. Tadpoles
derived from centrifugation experiments were treated similarly. Serial paraffin
sections, 5 fim thick, were stained with Mayer's hematoxylin and eosin. Primordial germ cells lodged in the genital ridges and dorsal mesentery were identified and counted on characteristics such as the large cell size, cytoplasm rich in
yolk platelets, and large, highly lobed, weakly staining nucleus.
To see the location of the 'germinal plasm', eggs at stages 2~, 2 and 3~ and
centrifuged eggs were fixed with 10 % formalin in 1/15 M phosphate buffer, pH
7-3. Fixed materials were sectioned serially parallel to the animal-vegetal axis
at thickness of 5 jam, and stained with Heidenhain's azan (Czolowska, 1969).
The distance from the vegetal surface to the centre of the islets of ' germinal
plasm' was measured, in every fifth section, parallel to the main axis of the egg,
under an ocular micrometer, x240 (minimum unit, 0-24 /tm). 50-200 islets
per egg were measured.
RESULTS
Although completely sterile tadpoles were rarely found in the normal populations, the average number of primordial germ cells per tadpole varied from
batch to batch and the actual number carried by an individual also varied
considerably within a batch.
U.v. irradiation with various doses
Stage 2~ eggs were irradiated with doses ranging from 750 to 6000 ergs/mm2
by varying irradiation time from 5 to 40 sec, respectively. The average number
of primordial germ cells of 10-30 tadpoles per dose is shown in Fig. 2, in
which it is seen clearly that the number of primordial germ cells decreases
along with the increase of the dose and that it reaches zero around 30-sec
irradiation.
Location of the 'germinal plasm' and the effect of u.v.
It is shown in Table 1 that 6000 ergs/mm2 of u.v., which drives all stage 2~
eggs into totally sterile tadpoles, drives greater proportions of stage 2 and 3 "
eggs only into partially sterile tadpoles. What is more, a greater number of
primordial germ cells are formed as the stage at irradiation advances.
Microscopic observations, on the other hand, of the interior of stage 2~ eggs
reveal that islets of 'germinal plasm' are located in the subcortical cytoplasm
of the vegetal hemisphere and are distributed uniformly around the vegetal pole
Primordial germ cells in Xenopus
0
(30)
5
10
15
20
25 30
(30) (30) (23) (30) (10) (30)
93
o40
(30)
Duration of irradiation (sec)
Fig. 2. Average number of primordial germ cells (PGCs) per tadpole, with standard
error. Eggs were irradiated at stage 2~ for various durations, and allowed to
develop into feeding tadpoles (stage 46). Dose rate was about 150 ergs/mm2/sec.
Number of tadpoles examined is given in parentheses. All eggs were obtained from
one batch.
Table 1. Location of the 'germinal plasm' at early cleavage stages and effect ofu.v.
irradiation on the formation of primordial germ cells (PGCs)*
Egg
Average distance
No. animals
of the islets r
Average no.
No. islets from the vegetal
With
PGCs per
surface (/*m)
Stage 8 Stage 46 germ cells
examined
tadpole
Stage 2995 (7)t
Stage 2
940 (9)
Stage 3 •
530 (9)
Un irradiated
3-3 ± 0-11
6-6 ±0-8
16-4 ±2-3
* All eggs were obtained from one batch.
t No. of eggs examined.
\ Mean value with standard error.
34
33
30
50
31
28
28
49
0
0
17 (60-7 %) 3-5±O-8J
25 (89-3 %) 7-9 + 11
49(100%) 33-5 ±6-2
94
K. TANABE AND M. KOTANI
Fig. 3. Location of the 'germinal plasm' at early cleavage stages. x270. Sections,
5/itn thick, were stained with Azan. (A) stage 2~; islets (arrows) of the 'germinal
plasm' are scattered in the vegetal subcortical cytoplasm. 9 islets are seen in this
figure. (B) stage 2; islets (arrows) are located more to the interior and nearer to the
cleavage furrow. (C) stage 3~; islets (arrows) are located farther from the egg surface
and coalesced along the cleavage furrow. (D) centrifuged egg at stage 2 ; islets
(arrows) are located much deeper in the cytoplasm. VP\ vegetal pole.
within an area of about a half of the diameter of the egg (Fig. 3 A). These islets
coalesce gradually during stages 2 and 3~ while at the same time migrating
interiorly along the cleavage furrow (Fig. 3B, C). The distances from the
vegetal surface to the centre of the islets are summarized in Table 1.
Centrifugation experiment
Histological observations of centrifuged eggs reveal that a considerable
number of islets of 'germinal plasm' has been displaced deeper into the cytoplasm (Fig. 3D). The average distances of the 'germinal plasm' from the
vegetal surface are shown in Table 2.
The number of primordial germ cells was counted in feeding tadpoles which
had descended from normal eggs, from eggs either irradiated or centrifuged
and from those irradiated immediately after centrifugation. The average number
of primordial germ cells per animal is shown in Table 2, which demonstrates
that tadpoles belonging to the last group possess a considerable number of
Primordial germ cells in Xenopus
95
Table 2. Location of the 'germinal plasm' after centrifugation and effect ofu.v.
irradiation on the formation of primordial germ cells (PGCs)*
Egg
No. islets
examined
Normal
1305 (9)t
—
Irradiated
Centrifuged
437 (8)
Irradiated after
—
centrifugation
Average distance
of the islets
from the vegetal
surface (/*m)
5-3 ± 0-6J
—
99-4 ±260
—
No. animals
f
Average no.
PGCs Dcr
tadpole
Stage 8
Stage 46
50
42
49
47
41
31
24-1 ±2-5
44
23
ll-5±2-2
51-1+9-3$
0
* All eggs were obtained from one batch.
t No. of eggs examined.
X Mean value with standard error.
Table 3. Effect of irradiating half the vegetal hemisphere on the formation of
primordial germ cells (PGCs)*
Area irradiated
No. of eggs No. of tadpoles
at stage 8
at stage 46
0 (Unirradiated)
, f Either one of the blastomeres
i
\ A half of both bl
blastomeres
45
49
45
42
42
42
Whole of the vegetal hemisphere
42
34
Average no. of PGCs
per tadpole
with standard error
31-8 ±4-9
151 ± 2 0
14-9 ±2-2
0
* All eggs were obtained from one batch.
primordial germ cells, in spite of the fact that they have received a dose strong
enough to cause complete sterility.
U.v. irradiation on known proportions of the vegetal hemisphere
Two series of experiments were performed to examine the relationship between
the amount of 'germinal plasm' and the number of primordial germ cells.
In the first experiment, a half of the vegetal hemisphere of early 2-cell eggs,
including either only one of the two blastomeres or a half of both, was subjected
to irradiation. The number of primordial germ cells found in the descendant
tadpoles is summarized in Table 3. It can be seen from these results that tadpoles
derived from either of the two groups carry nearly half the number of primordial germ cells possessed by the control animals. In the second experiment,
one to four quarters of the vegetal hemisphere of stage 2~ eggs were irradiated.
It is clearly seen in Fig. 4 that the average number of primordial germ cells
decreases in direct proportion to the increase of the area irradiated.
96
K. TANABE AND M. KOTANI
0
(37)
25
50
75
(37)
(33)
(40)
Area (%) of vegetal half irradiated
100
(36)
Fig. 4. Average number of primordial cells (PGCs) per tadpole, with standard
error. Different areas of the vegetal half were irradiated. Number of tadpoles
examined is given in parentheses.
DISCUSSION
Since more primordial germ cells are found in the descendant tadpoles as u.v.
dose decreases (Fig. 2) and u.v. hardly penetrates an anuran egg (Grant &
Wacaster, 1972), an apparent drop in sensitivity of an egg to u.v. associated
with the inward migration of the 'germinal plasm' during early cleavage stages
(Table 1) suggests that the islets of 'germinal plasm' located deeper in the
vegetal cytoplasm do not receive a sufficient dose to cause complete sterility
in tadpoles.
This is further supported by the results from the centrifugation experiments.
These show that tadpoles derived from irradiated eggs carry a considerable
number of primordial germ cells and that centrifuged eggs possess the 'germinal
plasm' located much deeper in the vegetal cytoplasm (Table 2). Hence it is
highly probable that the apparent drop of sensitivity to u.v. observed in eggs
beyond stage 2~ is not due to a gradual loss of sensitivity to u.v. of the 'germinal
plasm' with age, but to a decrease of dose reaching the 'germinal plasm'. It is
concluded from these evidences that a u.v.-sensitive germ-cell determinant is
present in the 'germinal plasm'.
It has been revealed by a recent study that the fine structure of the 'germinal
plasm' in Xenopus laevis is essentially the same as that of Ranapipiens (Czolowska, 1972). Comparison at ultrastructural and at light microscope levels of
Primordial germ cells in Xenopus
97
u.v.-irradiated and unirradiated 'germinal plasm' of Xenopus Jaevis has
demonstrated that a sterilizing dose (6000 ergs/mm2) of u.v. on the vegetal
hemisphere of stage 2~ eggs causes swelling and vacuolation of mitochondria
and fragmentation of electron-dense germinal granules without diminishing
the stainability in the histological sections (Ikenishi, Kotani & Tanabe,
1974). These facts suggest that the intactness of subcellular organelles such
as mitochondria and germinal granules is essential for differentiation of
primordial germ cells, and also support the conclusion from the centrifugation
experiments.
That u.v. irradiation of half the vegetal hemisphere reduced the average
number of primordial germ cells to nearly half may be interpreted as indicating
two features of the 'germinal plasm' (Table 3). One is that the irradiated 'germinal plasm' does not damage a certain function of the intact plasm in the formation of primordial germ cells, because the descendant tadpoles were able to
form primordial germ cells up to half the normal number. The other is that,
when such tadpoles are found to carry only half the normal number of
primordial germ cells, the intact plasm does not compensate for injured function
of the irradiated plasm. In other words, a definite amount (half) of the intact
'germinal plasm' is considered to be responsible for the formation of only a
limited number (half) of the primordial germ cells.
The latter suggestion is further confirmed by the result from u.v. irradiation
on known proportions of the vegetal hemisphere. The result - that the number
of primordial germ cells formed in tadpoles increases in direct proportion to the
increase of the area unirradiated - leads us to conclude that the number of
the primordial germ cell found in tadpoles is determined by the amount of
the u.v.-sensitive material(s) present in the 'germin alplasm'. This conclusion
justifies the suggestion that eggs bearing the reduced amount of 'germinal
plasm' grow into partially sterile tadpoles (Buehr & Blackler, 1970).
We are grateful to Drs Marina Dan Sohkawa and Mitsuru Furusawa for critical readings
of the manuscript, and Dr Seiichiro Kamisaka for his useful suggestions. Thanks are also
due to Mr Taizo Kimura for his help in photographic preparations.
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{Received 1 May 1973, revised 1 July 1973)