/. Embryo/, exp. Morph. Vol. 35, 3, pp. 617-623, 1976
Printed in Great Britain
617
Stage-sensitivity and dose-response
curve of u.v. effect on germ cell formation in
embryos of Xenopus laevis
ByKEN-ICHI IJIRI 1
From The Zoological Institute, University of Tokyo
SUMMARY
Ultraviolet light (u.v.) irradiation of the vegetal hemisphere of Xenopus laevis eggs resulted
in the elimination of primordial germ cells in tadpoles. Quantitative studies were performed
on this phenomenon. The stage sensitivity to u.v. inactivation of germ cell formation was
obtained for the early developmental stages ranging from immediately after fertilization to
small-cell blastula. It was found that u.v. irradiation at the stage immediately after fertilization was more injurious than irradiation at the beginning of the first cleavage. After deciding
optimal conditions for this u.v. irradiation experiment, a dose-response curve of the phenomenon was obtained. It showed a good agreement with the theoretical expectation, which
the authors had previously presented.
INTRODUCTION
Ultraviolet light (u.v.) irradiation of the vegetal hemisphere of many anuran
eggs results in the elimination of primordial germ cells in the tadpoles (Bounoure,
Aubry & Huck, 1954; Smith, 1966; Tanabe & Kotani, 1974; Ijiri & Egami,
1915a). This implies the existence of some u.v.-labile germ cell determinant in
the subcortical cytoplasm of the anuran egg.
Recently, Ijiri & Egami (1976) proposed a mathematical model for the germ
cell determination process. On the assumption that chance would distribute
this determinant in subsequently dividing cells during cleavage, a theoretical
equation which describes the relationship between survival of germ cells and
u.v. dose was presented. The purpose of the work reported here was (i) to
determine the stage-sensitivity curve for the early developmental stages ranging
from immediately after fertilization to small-cell blastula and (ii) to decide
optimal conditions for this u.v.-inactivation experiment in order to obtain a
clear dose-response curve for the phenomenon.
1
Author's address: Zoological Institute, Faculty of Science, University of Tokyo, Hongo,
Tokyo 113, Japan.
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K. IJIRI
MATERIALS AND METHODS
Collection of eggs
Fertilized eggs of Xenopus laevis were obtained by injecting human chorionic
gonadotrophic hormone into a pair of mature toads (Gurdon, 1966). Stimulated
toads were kept in a container which had two compartments with a shutter
separating these compartments. Each compartment was lined with a plastic
meshwork basket which was kept out of contact with the floor of the container.
In order to obtain eggs with clean jelly coats for u.v. irradiation, only those
eggs clinging to the bars of the mesh were collected.
After an hour of laying, the eggs were collected at 10 min intervals, each time
transferring the toads into the other compartment. In this way, synchronous
development, at least during the early cleavage stages, was obtained. Collected
eggs were either immediately irradiated or left to incubate at 20 °C for future
irradiation. Embryos were staged according to the normal table of Nieuwkoop
& Faber (1956) (N. & F.).
U.v. irradiation
Eggs, with intact jelly coats, were pipetted into a quartz dish and oriented
with the animal hemisphere upwards. Excess water was then removed from the
dishes as completely as possible. By this removal of water, all the eggs were
pressed against the bottom of the dish with their own weight, having the vegetal
hemispheres somewhat flattened.
Irradiation of the vegetal hemispheres was performed by placing the quartz
dish horizontally over the u.v. source. A low-pressure mercury lamp emitting
nearly 95 % of its u.v. energy at a wavelength of 253-7 nm was used as the u.v.
source. The dose rate reaching the vegetal surface of the eggs was measured by a
u.v. dosimeter (UVR-254, TOPCON). After irradiation, eggs were placed in
glass vessels with stored tap water to develop at 20 °C.
Rearing tadpoles and fixation
To minimize fluctuations in the rate of tadpole development, the following
procedure was undertaken. Animals were reared at 20 ± 1 °C, about 20 tadpoles in
a litre of water, and were fed daily on the diet for goldfish from stage 45 onwards.
It has been supposed that there is a period of mitotic arrest between the initial
segregation of primordial germ cells and shortly after their arrival at the gonadal
area (Bounoure, 1925; Cheng, 1932; Blackler, 1970). From our previous result
of germ-cell counting during normal development of the tadpole at 20 °C
(Ijiri & Egami, 19756), the stages up to 47 seem to correspond to such a period
of mitotically dormant state, and probably exhibit the number of primordial
germ cells initially formed.
Throughout the present experiments, tadpoles were fixed in Bouin's solution
at stage 47", serially sectioned transversely in paraffin at 7 [im thickness, and
U.v. effect on germ cell formation in Xenopus
(10)
619
(9)
(10)
£ 10
(10)
(10)
(10)
0-5
(10)
(9)
00)
Stage of u.v. irradiation
i
i
30-40 50-60 Early Mid 2 cell 4 cell
min
min 2~ to
'
late 2~
After fertilization
0-10
min
i
8 cell 16-32
cell
Fine-cell
blastula
Large-cell
Control
blastula
(unirradiated',
Fig. 1. Stage sensitivity for the early developmental stages. Eggs and embryos were
irradiated at the dose rate of 137 ergs/mm2/sec. Germ-cell counting was performed at
stage47~. Number of tadpoles examined is given in parentheses. Survival of germ cells
has been expressed with standard error. O, Data obtained from one batch of eggs
irradiated at the u.v. dose of 4110 ergs/mm2. Average number of germ cells of control (unirradiated) tadpoles was 22-8 ± 14 (S.E.). • , Data obtained from one batch
of eggs irradiated at the dose of 1370 ergs/mm2. Average number of germ cells of
control tadpoles was 33-4±2-1 (S.E.).
stained with Delafield's hematoxylin and eosin. Direct germ-cell counting was
performed by tracing all the germ cells through serial sections from the cloacal
position rostrally until the genital ridges disappeared.
RESULTS
Stage sensitivity to u.v. irradiation
The stage sensitivity to u.v. inactivation of germ cell formation was obtained
for the early developmental stages ranging from 0-10 min after fertilization to
the small-cell blastula stage (Fig. 1). For convenience, the number of germ cells is
presented in terms of survival rate, considering the number of germ cells in
unirradiated controls as 1-0.
A u.v. dose of 4110 ergs/mm2 given to the eggs 0-10 min after fertilization
resulted in complete elimination of primordial germ cells. However, the same
dose applied at subsequent developmental stages resulted only in partial
sterility. As the cleavage proceeds, the effect of u.v. on germ cell formation
decreases. After the 16-32 cell stage, u.v. irradiation had no observable effect
on germ cell formation, such animals exhibiting the same number of germ cells
as unirradiated controls.
620
K. IJIRI
A
10 -
30-
20-
Z
3 0-
10-
001 -
1000 2000 3000 4000 5000 6000 7000
Total u.v. dose (ergs/mm2)
0
1000 2000 3000 4000 5000 6000
Total u.v. dose (ergs/mm2)
Fig. 2. Dose-response curve. Eggs were irradiated at 30-40 min after fertilization,
and germ-cell counting was performed at stage 47~. All eggs were obtained from one
batch. V, Dose rate of 42 ergs/mm2/sec; O, dose rate of .137 ergs/mm2/sec. (A)
Average number of primordial germ cells per tadpole versus the incident total dose,
with standard error. (B) Same data expressed in a semi-log plot.
One interpretation of the decrease in u.v. sensitivity after the first division is
that the germinal determinant moves into the interior of the egg. Thus, it
becomes less accessible to the injurious effects of u.v. irradiation. This idea,
connected with the apparent movement of microscopically identifiable cytoplasm termed 'germinal plasm', has been shared by many authors (Bounoure
et al. 1954; Smith, 1966).
In both u.v. irradiation experiments of 4110 ergs/mm2 and 1370 ergs/mm2,
the highest sensitivity immediately after fertilization was confirmed. This result
does not agree with the previous reports on Rana temporaria eggs (Bounoure
et al. 1954) and Rana pipiens eggs (Smith, 1966). This difference is considered
in the Discussion.
The stage of eggs 30-40 min after fertilization has been taken as the optimal
stage for u.v. irradiation of Xenopus laevis eggs, since there exists no drastic
change in the sensitivity curve around this stage.
U.v. effect on germ cell formation in Xenopus
621
Dose-response relationship curve
A dose-response curve was established after irradiating the vegetal hemisphere of eggs at the stage of 30-40 min after fertilization and counting the germ
cell number in the tadpoles at stage 47~. The results are expressed in Fig. 2, A,
B. Their characteristics can be summarized as follows. (1) The number of germ
cells takes the value 0 for u.v. doses beyond a certain value (5500 ergs/mm2).
(2) As shown in the semi-log plot of Fig. 2B, for the small dose range (0-1500
ergs/mm2), the curve has no shoulder and exhibits almost a straight line of a
certain slope and (3) as the u.v. dose becomes greater than this range, the slope
becomes steeper and steeper.
Another experiment was performed on a different batch of eggs, irradiating
at the early 2~ stage. This stage corresponds to the beginning of the first cleavage
division and has been adopted by most authors in their u.v.-irradiation experiments (Bounoure et al. 1954; Smith, 1966; Tanabe & Kotani, 1974). The same
characteristics mentioned above were again observed in this experiment.
DISCUSSION
The results of the present experiments, based on quantitative studies, demonstrate the u.v.-inactivation phenomenon of germ cell formation in Xenopus
laevis embryos. A stage-sensitivity curve of this phenomenon has been obtained
during early embryonic development, with special attention being paid to the
sensitivity during the period between immediately after fertilization and the first
cleavage division. No work has yet been reported on the effectiveness of u.v.
on the eggs immediately after fertilization in any anuran species. The present
experimental results reveal that u.v. irradiation was effective even at such a
stage, implying the existence of a u.v.-labile germ cell determinant in the eggs
immediately after fertilization. The stage of 0-10 min after fertilization exhibited
the highest sensitivity. Ultraviolet light inactivation at the stages 0-60 min after
fertilization was more injurious than was irradiation at the beginning of the
first cleavage. This result does not agree with the previous reports on two other
anuran species.
Smith (1966), in his u.v.-inactivation experiments on Rana pipiens eggs,
reported that the stage of 30 min before the first cleavage division (i.e. 2-5 h
after fertilization) was half as sensitive as the stage of the beginning of the first
cleavage division. In Rana temporaria, Bounoure et al. (1954) also reported that
the stage just before the first cleavage division was more sensitive than the stage
of 1-5-2-0 h after fertilization. According to them, the germ cell determinant
is at first spread over a rather extensive area of the vegetal hemisphere, and at
the beginning of the first division it becomes concentrated so that u.v. can
effectively damage it.
In our experiments, we removed water completely from the quartz dish, so
622
K. I J I R I
that the eggs were pressed against the bottom of the dish with their own weight.
In Xenopus eggs, a large area of the vegetal hemisphere could be flattened by this
method. For Rana eggs, both the above authors compressed the eggs with a lid.
One explanation for the above disagreement could be the possibility that, since
Xenopus eggs are softer than Rana eggs, a larger area is flattened and thus more
germ cell determinant is successfully irradiated. The histological observations
also seem to show that in Xenopus islets of germinal plasm stained blue in the
eggs of 50-60 min after fertilization are situated nearer to the cell membrane
than at the 2~ stage. These facts may account for the results of our present
experiments.
Ijiri & Egami (1976) presented a mathematical model for the germ cell
determination process. This model supposes that at a certain critical period, the
germ cell determinant has some interaction with the nucleus of a cell, and the
amount of this interaction will decide whether this cell differentiates into a germ
cell or a somatic cell. They assumed that the germ cell determinant will be
partitioned in subsequently dividing cells during the cleavage, following the
probabilistic rule governed only by the geometric relationship between the area
of the cell surface and the amount of germ cell determinant contained in the
cell. To connect the macro dose-response curve based on germ-cell counts with
events at the molecular level, they introduced the idea that, at the molecular
level, damage to the germ cell determinant obeys the hit theory with respect to
u.v. dose. According to this model, they wrote mathematical equations which
described the relationship between the survival of germ cells S and the u.v. dose
D, presenting
P-2<TD
_
n
S = 0 (Do< D),
as a final formula. Here cr is a constant termed 'molecular inactivation cross
section' of germ cell determinant. C and Do are also other constants (for details,
see Ijiri & Egami, 1976).
The general characteristics of the dose-response curve predicted by this
equation are as follows. (1) Survival S takes the value 0 for the value of D
beyond a certain dose Do. (2) For small values of D, because of the term e~2<rD,
the curve expressed in semi-log fashion exhibits an almost straight line of slope
2cr. (3) However, as D increases, the slope becomes steeper and steeper owing
to the presence of the constant C.
The dose-response curves in Fig. 2 show good agreement with these theoretical expectations, and thus support this probabilistic model of the germ cell
determination process.
It is possible to obtain from the dose-response curve the value of molecular
inactivation cross section cr of the germ cell determinant, which value may offer
access to its biochemical nature. However, for this, dose D must be calculated
U.v. effect on germ cell formation in Xenopus
623
as the dose actually arriving at the germ cell determinant. Measurement of the
transmittance of the vegetal subcortical plasm of Xenopus laevis egg and the
depth of germinal plasm from the egg surface at the stage of u.v. irradiation is
now an urgent necessity.
The author wishes to thank Professor Nobuo Egami, Zoological Institute, University of
Tokyo, for his constant guidance and valuable discussions. Also, thanks are offered to
Professor P. N. Srivastava, Department of Life Science, J. Nehru University, India, for his
critical reading of this manuscript.
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{Received December 1975; revised 5 February 1976)