Protein Synthesis During the First Three Cleavages in Pond Snail Eggs
(Lym n aea StagnalisJ
B r ig it t e J o c k u s c h *
Max-Planck-Institut für Meeresbiologie, Tübingen, Abt. Bauer
(Z . Naturforschg. 23 b, 1512— 1516 [1968] ; eingegangen am 7. M ai 1968)
Egg cells of the pond snail Lymnaea stagnalis were isolated and reared in an inorganic medium,
in which they readied the blastula stage with the same rate and yield as in the egg capsules. Syn­
chrony of cell division was 70 — 90% depending on temperature and number of cell division.
3H-leucine incorporation into hot TCA-insoluble material as a function of developmental stage was
studied by autoradiography. The rate of leucine incorporation during the metaphase of the first
three cell divisions was found to be ca. 30% of the incorporation rate during the corresponding
interphases. The interphase rates increased from a very low level before nuclear fusion to a 25
fold value in the third interphase. 3H-leucine incorporation could be inhibited by 78% by 5 x 10“ 3 m
puromycin and hence seems to represent protein synthesis.
T im e -c o u rs e s tu d ie s o f in c o r p o r a t io n o f la b e le d
w ork pond snail eggs have been used. In contrast to
a m in o a c id s , to d e te rm in e w h e th e r the p ro te in s y n th e ­
sea urchin eggs, they belong to the mosaic type of
s is o f the c e ll is d e p e n d e n t o n the m ito tic c y c le o r n o t,
egg and have spiral cleavage. There is an additional
h a v e b e e n c a r r ie d o u t b y s e v e ra l w o rk e rs . M a n y o f
reason fo r studying snail eggs in this manner. F er­
th e m
tilization and nuclear fusion are temporally separated
(e . g. C a r n e ir o a n d L e b l o n d 1, P r e s c o t t a n d
B e n d e r 2, B a s e r g a 3, K
land
4, J o hnso n a n d H
o nrad
o l­
5, S a l b a n d M a r k u s 6) h a v e s tu d ie d m a m m a lia n
tissu e
c u ltu re
c e lls ,
( B ootsm a, B u d k e
w h ic h
can
and V o s 7)
be
in some species and thus protein synthesis between
these two events can be analyzed.
s y n c h ro n iz e d
to o b ta in s u ffic ie n t
M aterial and Methods
a m o u n ts o f c e lls in the sam e m ito tic stage. T h is typ e
o f c e ll is d iffe re n tia te d , c o n ta in s o n ly s m a ll p o o ls o f
a m in o a c id s a n d need s a c o m p a ra tiv e ly lo n g p e rio d
b e tw ee n the m ito tic c y c le s
( T a y l o r 8) . I n c o n tra s t
the egg c e lls s u p p lie d w ith y o lk p ro te in s u n d e rg o
m ito tis e v e ry fe w h o u rs . In v e s tig a t io n s h a v e b e e n
p e rfo rm e d w ith sea u rc h in eggs
I m m e r s 10, H
u l t in
(e. g. K
11, G r o s s 12, G ro ss
avanau
and
Fr
y
9,
13,
S o f e r , G e o r g e a n d I v e r s o n 14) w h ic h a re o b ta in a b le
in la rg e a m o u n ts a n d c a n be e a s ily s y n c h ro n iz e d b y
c o n t r o llin g the p o in t o f f e r t iliz a t io n . T h e sea u r c h in
egg ,
h o w e v e r, b e lo n g s to the re g u la t o r y
ty p e
of
e g g c e ll a n d s tu d ie s o n p ro te in s y n th e s is s h o u ld be
e xte n d e d to o th e r typ es o f egg s. F o r the p re se n t
* Present address: McArdle Laboratory for Cancer Research,
University of Wisconsin, Madison, Wisconsin, 53706.
1 J. C a r n e ir o and C. P. L e b lo n d , Science [Washington] 129,
391 [1959].
2 D. M. P r e s c o t t and M. A. B e n d e r, Exp. Cell Res. 26, 260
[1962].
3 R. B a s e r g a , Biochim. Biophysica Acta [Amsterdam] 61,
445 [1962].
4 C. G. K o n r a d , J. Cell Biol. 19, 267 [1963].
5 T. C. Johnson and J. J. H o lla n d , J. Cell Biol. 27, 565
[1965].
6 J. M . S a lb and P. J. M a r c u s , Proc. nat. Acad. Sei. U S A
54, 1353 [1965].
Preparation and growth characteristics of the egg
cells. Eggs from the pond snail Lymnaea stagnalis were
used. The snails were kept in tanks with fresh water
at 17 °C and fed with lettuce. To obtain large quanti­
ties of egg masses temperature in the tanks was raised
to 25 °C. By this change of temperature the snails were
stimulated to lay their eggs ( R a v e n 1S) on the lower
side of floating petri dishes. The egg cells could be
isolated by rolling the egg capsules out of the outer
jelly on a filter paper and pricking the egg capsules
with a needle ( R a v e n and M i g h o r s t 16) . Because the
eggs in the front part of the egg masses, which were
laid about 10 min earlier than the last ones, begin
their development earlier, the first and the last 10 eggs
of each egg mass were rejected, so that from each egg
mass 50 —60 egg cells were obtained. The egg cells
7 D. Bootsm a, C. B u dk e and O. Vos, Exp. Cell Res. 33, 301
[1964].
8 E. T a y lo r , J. Cell Biol. 19, 1 [1963].
9 J. L. K a v a n a u , Exp. Cell Res. 7, 530 [1954].
10 J. Immers, Exp. Cell Res. 18, 585 [1959].
11 T . H u lt in , Exp. Cell Res. 3, 494 [1951].
12 P. R. G ro s s , J. exp. Zoology 157, 21 [1964].
13 P. R. G ro s s and B. J. F r y , Science [Washington] 153, 749
[1966],
14 W . H. S o f e r , J. F. G e o r g e , and R. M. Iv e rson , Science
[Washington] 153, 1644 [1966].
15 C h r . P. R a v e n , J. Embryol. exp. Morph. 12, 805 [1964].
16 C h r . P. R a v e n and J. C . A. M ig h o r s t , Proc., Kon. nederl.
Akad. Wetensch. 59/9, 3 [1946].
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in metaphase
% cells
from very fresh egg masses are fertilized but have to 100
complete maturation with the extrusion of the first and A
second polar bodies before fusion of the two pro- | gQ
nuclei takes place ( R a v e n 17) . The freed egg cells were 8
put into a medium containing 0.60 g NaCl, 0.42 KC1,
0 .2 4 g CaCl2 per 1 00 0 ml, buffered with m /50 Tris
60
(hydroxymethyl)-aminomethane-HCl at pH 6.7. In this *=
medium the development reached the gastrula stage, 5 40
where the embryos died. Death at this stage may be =§
explained by the observation that the gastrulae begin ^ 2Q
to grow, consuming the protein-containing fluid of the 0
egg capsules ( E l b e r s and B l u e m in k 18) . Mitotic stages
were identified after fixation with ethanol acetic acid
540
360
420
480
1:1 and staining with orcein acetic acid. Within the
Time (m in ) —
first three cleavages the time course is identical in egg
Fig. 2. Relation between developmental temperature and
cells in their natural environment (the controls) as well
as in naked egg cells in the medium (Fig. 1). The cor­ generation time. A — A : cells developing at 13 °C in culture
medium. • — • : cells developing at 20 °C . 0 — 0 : the same
relation between duration of cell division and tem­
at 27°. High synchronization is correlated with low tempera­
perature and between synchronization and number of
ture and decreases with number of cleavage. Time counting
cell divisions are presented in Fig. 2. A ll experiments
starts with nuclear fusion = 0 min.
were carried out at 20 °C. The time-course diagram
allowed the determination of the desired mitotic phase
way (Kodak Stripping Plates, directions for use) to
of the living cells.
ensure adhesion of the stripping film. To obtain suffi­
cient flat preparations for minimal self absorption, the
mounted cells were squeezed by low pressure on coverslips and frozen with dry ice. The thickness of the
samples was calculated from volume of fixed cells and
increase of the areas by squeezing. The values obtained
were from 1.0 to 1.5 /um thickness. After lifting off the
coverslips with a razor blade and airdrying, the slides
were rinsed twice in 50% alcohol, once in distilled HaO
and extracted with 5% trichloracetic acid (TC A) at
90°C for 30 min, to remove unincorporated precursor.
After removal of TCA the slides were coated with strip­
180 210 240 270 300 330
ping film (Kodak AR 10) and exposed for 20 days at
Time (min a fter nuclear fusion) — »
5 — 7 °C. Grains were counted under a phase contrast
Pj
M; A] +Tj J;
P2
1^2 A 2 +T2
O2
P3
A3 +T3 J j
microscope. The background, which never exceeded
16% of the counted grains over the cell, was subFig. 1. Synchronization and conformity of cleaving eggs
stracted.
during the first three cell cycles in various media. (J : cells in
Incorporation of zH-leucine before nuclear fusion
their natural jelly and capsule fluid (egg album en). A : cells
and during the first cleavages. Kinetic experiments were
without outer jelly but within the capsules. • : cells freed of
carried out by measuring the incorporation over a range
both, and put into culture medium. In the lower part the dura­
tion of the various phases during mitosis is shown. P 1(2>3:
from 0.5 —30 min before nuclear fusion, during the first
1st, 2nd, 3rd prophase, M : metaphase, A + T : ana- and telo­
metaphase, and during the first interphase stages. At the
phase, I: interphase stages. Temperature: 20 °C .
onset of each desired stage 30 //C/ml of 3H-leucine were
added. Samples were fixed after an exposure time up
to 30 min, longer exposures not being possible because
Autoradiographic techniques. The cells were ex­
posed to 10 —30//C/ml of L-leucine-4,5-T (The Radio­ of the beginning of the next stage. In the experiments
concerning the incorporation during the first three
chemical Centre Amersham 1 mC/mMol). At 20 °C the
metaphase stage lasts about 27 —30 min (see Fig. 1),
meta- and interphases an incubation with 10 //C/ml for
so that a pulse of 20 min ensured a good coincidence
20 min proved adaequate to give countable grain num­
with the considered mitotic stage. At the end of incuba­ bers per cell.
Uptake kinetics for 3H-leucine by the cells. In order
tion, cells were fixed (four changes) and kept in etha­
nol acetic acid 1:1 for 12 hrs at 6 °C. Then they were
to exclude the possibility that the differences in grain
rinsed with a 3:1 ethanol acetic acid mixture and trans­ counts reflect merely different diffusion times ( M it c h i ferred to slides which had been pretreated in the usual
so n and C u m m in s 19) the uptake speed was measured.
17 C h r .
P. R aven , Morphogenesis: The analysis of molluscan
development, Pergamon Press, London 1966.
18 P. F. E l b e r s and J. G. B lu e m in k , Exp. Cell Res. 21, 619
[I9 6 0 ].
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After an exposure to 10 /^C/ml of 3H-leucine of up to
5 min, the living cells were, very quickly, washed
twice in the culture medium at 0 °C and killed by
freezing with dry ice. The above described procedures
were followed here too. Grain counts in this case were
made after exposing the stripping film for only
8 days. After 5 min of incubation with 3H-leucine, meta­
phase and interphase cells had taken up the same quan­
tities of radioactive amino acid and had the same
(estimated) numbers of grains per cell (about 12 fold
higher than the highest value measured in incorpora­
tion experiments). This was obviously due to the much
higher quantity of non-incorporated precursor, which
had not been removed by TCA, as in the experiments
concerning incorporation into cell proteins.
Inhibition of incorporation by puromycin. Over a
range from 5 x IO-3 to 1 x 10~6m puromycin the inhi­
bition of incorporation in the first interphase was
measured. Cells were preincubated with the inhibitor
immediately after cleavage and 20 min later 30 ^aC/ml
3H-leucine were added for a pulse of 20 min. Fixa­
tion and further treatment were the same as described
above, the samples were exposed for 20 days.
the following anaphase. As the diffusion tests (see
Material and Methods) indicated, these results are
not due to different uptake times of interphase and
Results
The grain count comparisons for interphase and
metaphase cells after an exposure from 0.5 — 30 min
show a much higher incorporation in the first inter­
phase ( I x) than in metaphase (M i). A t the end of
the incubation time (30 min point in Fig. 3 ), the
Prophase
Anaphase
~20
~30
Time (m i n ) ------=
Fig. 3. Kinetics of incorporation of 30 /iC/ml 3H-leucine
during sperm nucleus migration before nuclear fusion (S M ),
in first metaphase (M t) and first interphase ( I j ) . Arrows indi­
cate the beginning of the stated next mitotic stage. The 30 min
average values are given with standard errors of the mean
(vertical sections).
metaphase grain numbers were only 30% o f the
interphase values. Fig. 3 shows the linear ascent
during the first 20 min of incubation in Ij^ fo l­
lowed by a noticeable flattening shortly before the
next prophase, the same slope is also present at the
end of the first metaphase (M x) incorporation before
Fig. 4. Autoradiographs of whole two-cell stage in the inter­
phase (a ), cutting of same areas of the first metaphase (b)
and the first interphase ( c ) . Exposure to 30 ,uC/ml 3H-leucine
for 20 min. Cells were squeezed to a maximum thickness of
1.0 — 1.5 jum. Differences can be observed only in the number
of grains; no distinction in the regions of interphase nuclei
(Fig. 4 c, above) or metaphase spindle apparatus (Fig. 4 b, in
the centre) can be seen.
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metaphase cells; one can attribute these differences
to protein synthesis. This synthesis, measured by in­
corporation of radioactive amino acid before nu­
clear fusion but after fertilization, is given in the
third graph (S M ) of Fig. 3. At 20 °C migration of
the sperm nucleus, which begins after extrusion of
the second polar body, takes 30 min. In this time
less than 30% incorporation is found, compared to
the first metaphase stage.
The autoradiographs show a homogenous distri­
bution of labeling over both macro- and micromeres
in the third interphase ( I 3) after the separation of
the first quartette of micromeres.
Incorporation o f 3H-leucine can be inhibited by
relatively high concentrations o f puromycin (Fig. 6).
By preincubating with the inhibitor for 20 min be­
fore adding the amino acid, the effect can first be
noticed with 1 x 10-4 m puromycin in I x . With a
As shown in Fig. 4 incorporation after 20 min
incubation in interphase and metaphase differs only
with respect to the grain number per cell. In both
cases there was uniform distribution of the grains.
No significant differences in the regions of the inter­
phase nuclei or metaphase spindle apparatus were
visible.
The results on protein synthesis during the first
three mitotic cycles are shown in Fig. 5. Total in­
corporation increased rapidly. Each interphase stage
Log molarity o f Puromycin — *■
Fig. 6. Effect of puromycin on protein sythesis during the
first interphase. The inhibitor had been given in each sample
20 min before adding 30 /uC/ml 3H-leucine for 20 min. Verti­
cal sections indicate standard errors of the mean.
concentration o f 5 x 10_ 3 m puromycin normal pro­
tein synthesis is reduced to 22%, but cells are able
to cleave within normal times up to the four-cell
stage before being blocked ( I 2). Further increase of
concentration leads to an atypical appearance of the
cells, so that further decrease of grain counts may be
due to unspecific effects.
120
180
Time (min)
Fig. 5. Relations in total protein synthesis of the first three
cell cycles to mitotic phases. Exposure to 10 /uC/ml sH-leucine
for 20 min. S M : incorporation during sperm nucleus migra­
tion, N F : nuclear fusion,
: first metaphase, I t : first inter­
phase. O — O: percentage of cells in metaphase.
incorporated a considerably higher percentage of
precursor than the last one. The same was the case
in the metaphase stages. The decrease o f synthesis
during each metaphase following an interphase is
maintained so that metaphase incorporation in the
first three mitotic cycles is always about 30% of the
incorporation of the following interphase. The pro­
tein synthesis in the time of sperm nucleus migration
before nuclear fusion was also very low ; the rela­
tion between this value and the values of first meta­
phase and interphase is the same as in Fig. 3.
Discussion
The kinetic studies before nuclear fusion and in the
first metaphase and interphase (Fig. 3) show clearly
a great increase of protein synthesis up to this last
stage, or, less probably, a corresponding reduction
of the size o f the leucine pool. The fact of the very
low level of incorporation before nuclear fusion
suggests that not only fertilization, but nuclear fu­
sion itself, triggers the increase of protein synthesis,
although in this case one has to assume a value for
protein synthesis no higher before fertilization than
after it. The latter could not be ascertained in this
study, because fertilization takes place within the
snails. On the other hand there is no normal inter­
phase cell after nuclear fusion [£. e ., spindle rem­
nants exist up to the first prophase (R av en 17) ]
so that the cell cannot produce as much protein as in
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the first interphase. The decrease of the rate o f in­
corporation in late M t (Fig. 3) may indicate
a minimum of protein synthesis in the ana­
phase or telophase cell. Over a pulse range of
5 — 30 min, the stated difference of incorporation,
depending on the mitotic stages, cannot be due to
different diffusion times, as the diffusion test shows
the same values after only 5 min incubation with
3H-leucine for the studied meta- and interphases.
There may be however, the possibility of rhythmical
changes in the amounts of leucine in the amino acid
pools o f the cell between meta- and interphase. As
is shown in Fig. 5 the protein synthesis during the
first three cell cycles rises rapidly and up to the
formation of the first quartette of micromeres within
the third cleavage there is no indication that protein
synthesis may reach a constant level. According to
M o r r i l l 20 the protein content of the cleaving egg
and the young embryo of Lymnaea palustris does
not change until the second day. Assuming this to
be the same in Lymnaea stagnalis, the increasing
rate o f protein synthesis during the first three cell
cycles should be only a transformation o f yolk pro­
teins and not net synthesis. Whereas G r o s s and F r y 13
found that protein synthesis in the sea urchin egg did
not diminish during the first and second cell cycle,
S o f e r , G e o rg e and I v e r s o n 14 found a distinct de­
crease of protein synthesis after early prophase in
sea urchin eggs. The discrepancy between these two
results has not yet been resolved, but the second one
agrees with the present experiments in snail eggs and
also with the data of P r e s c o t t and B e n d e r 2 on
Chinese hamster cells, where the rate of incorporation
o f amino acids falls to 25% o f the average interphase
level during telophase. It would give an interesting
new aspect to this problem to investigate whether in
egg cells such as molluscs or sea urchins the poly­
ribosomes also disappear during mitosis as they do
in HeLa cells ( S c h a r f f and R o b b in s 21) .
20 J. B. M o r r i l l , Acta enbryol. Morphol. Exper. 7,131 [1964].
21 M. D. S c h a r f f and E. R obbins, Science [Washington]
992 [1966].
The author wishes to express her gratitude to D r.
W. K e y l and Mr. H a g e le fo r technical ad vice, to Mr.
F r e ib e r g fo r the draw in g s, to Prof. D r. H. B a u e r and
D r. G. C z ih a k fo r d iscu ssin g the m a n u scrip t. T h is w o rk
w as c a rrie d out in D r. C z ih a k ’s g roup as p a rt of the
p ro g ra m “ B io ch e m ica l E m b ry o lo g y ” and w as supported
b y “ Deutsche F orsch u ngsg em einschaft” .
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151,