/ . Embryol. exp. Morph. Vol. 26, 3, pp. 351-366, 1971
Printed in Great Britain
351
Timing of the phases of the cell
cycle with tritiated thymidine and Feulgen
cytophotometry during the period of synchronous
division in Lymnaea
By J. A. M. VAN DEN BIGGELAAR 1
From the Zoological Laboratory, University of Utrecht
SUMMARY
The duration of the phases of the cell cycle during the 1-, the 2- and the 4-cell stage of the
Lymnaea egg were determined with [3H]thymidine and with Feulgen cytophotometry. The M,
S and G2 phases occupy 48, 27 and 25 % of the first three cell cycles. A Gx phase cannot be
observed. Only from the 4-cell stage was [3H]thymidine readily incorporated into DNA. The
theory that an increase in respiration during the S phase of the 4-cell stage is connected with
the energy requirements of DNA synthesis is discussed.
INTRODUCTION
In eggs of Lymnaea stagnalis it has been observed that cleavage delay and
alterations of normal development, induced experimentally during the first
cleavages, are related to periodically recurring cell activities (Geilenkirchen,
1964, 1966; Carney & Geilenkirchen, 1970; Labordus, 1970a, b, c; E. K. BoonNiermeyer, personal communication). Depending on the kind of treatment,
various sensitive periods have been demonstrated within each cleavage cycle.
For the interpretation of the results information had to be obtained about
recurring metabolic processes during the early period of development.
Reduplication of DNA in each cleavage cycle represents one of these processes. In the study of DNA metabolism the incorporation of [3H]thymidine is
frequently used as an indication for DNA synthesis. Another much-applied
technique is the microphotometric determination of the Feulgen dye content of
the nuclei. Results with both techniques will be dealt with in this paper.
1
Author's address: Zoologisch Laboratorium der Rijksuniversiteit, Utrecht, Janskerkhof 3,
The Netherlands.
352
J. A. M. VAN DEN BIGGELAAR
MATERIALS, METHODS AND RESULTS
Timing of the phases of the cell cycle using [3H]thymidine and
autoradiography
Introduction
DNA synthesis can be demonstrated by the incorporation of labelled precursors. If the incorporation of thymidine (Tdr) is used as an indication of
DNA synthesis, the following must be taken into consideration.
Tdr has to be converted successively into thymidine monophosphate (dTMP),
thymidine diphosphate (dTDP) and thymidine triphosphate (dTTP) before it
can be incorporated into DNA. This pathway is a minor route, it is a salvage
pathway (Cleaver, 1967). The major route starts with the methylation of
deoxyuridine monophosphate into dTMP (Friedkin, 1963). For this reason
incorporation of Tdr can only be used as a reliable indicator of DNA synthesis
if the enzymes for conversion of Tdr into dTTP are present. Even if the enzymes
are present it must be realized (a) that exogenous Tdr may exert a feedback
control upon the synthesis of thymidine nucleotides in the cell (Nemer, 1962),
(b) that high concentrations of Tdr in the medium may block the cells from
entering the DNA synthetic phase (Bootsma, Budke & Vos, 1964), and (c) that
cells which fail to incorporate Tdr may replicate DNA (Stone & Prescott, 1964).
Evidently, the rate of incorporation of Tdr does not necesssarily reflect the rate of
DNA synthesis, and lack of detectable incorporation does not necessarily
indicate absence of DNA synthesis.
Materials and methods
Egg masses oiLymnaea stagnalis were obtained by spontaneous or stimulated
oviposition. Stimulation was achieved by refreshing and aerating the water of
the aquaria in which the adult snails were kept and raising the temperature to
25 °C (Raven & Bretschneider, 1942). The egg capsules were freed from an egg
mass by cutting it open longitudinally and rolling the capsules over moistened
filter paper. As the sticky mucus adheres to the paper the capsules become
detached from each other. The eggs were stored in salt dishes with copper-free
tap-water until used for experiments.
Within one and the same egg mass the divisions are not synchronous. Groups
of almost synchronously dividing eggs were obtained by selecting eggs which
started to divide within successive periods of 2 or 3 min. The first and last group
of each egg mass were used to determine the average duration of the successive
cleavage stages. The mean time-lapse between the first and last dividing group
was about 30 min. The temperature at which the experiments were performed
varied from 22 to 26 °C.
Autoradiography. The concentration of [3H]Tdr (Schwarz, specific activity
3 Ci/mM) in which the eggs were incubated was 25 fid per ml tap-water. After
incubation the eggs were decapsulated with watchmaker's forceps, fixed for at
Timing of the phases of the cell cycle in Lymnaea. /
353
least 1 h in alcohol 96 %, acetic acid 3:1, and washed in alcohol 70 % for one to
several hours. Then the eggs were transferred to a 2% agar solution at 60 °C in
which they were oriented. After solidification, agar containing eggs was cut into
small pieces which were dehydrated in an alcohol-amyl acetate series. After embedding in paraffin the blocks of agar were sectioned at 2/*m thickness. Sections
were mounted on coated slides and covered with Kodak ARlOstrippingfilm. In two
experiments a number of slides were previously treated with DNase (Nutritional
Biochemical Corporation) for 3 h at 37 °C. The enzyme was dissolved at a
concentration of 0-1 /^g/ml in a 0-05 M Tris-buffer (pH 7-4) with a final concentration of 0-003 M-MgClg. The slides were stored at 5 °C in light-tight boxes
with silica gel. After an exposure time of 2-8 months the autoradiographs were
developed in Kodak D-\9b developer for 5 min at 18 °C, rinsed for \ min in
distilled water, fixed in acid fixative for 15 min and finally washed in filtered tapwater for 30 min. After drying, the autoradiographs were stained with methyl
green-pyronine. A nucleus was scored as labelled if the total number of grains
over the sections thereof was significantly higher than over comparable surfaces
of the film adjacent to the egg.
Cytophotometry. At successive intervals eggs were decapsulated, fixed for 1 h
in a mixture of alcohol 80 %, formol 40 %, acetic acid (85:5:10) or in alcohol
96 %, acetic acid (3:1), and subsequently washed in alcohol 96 % for about 24 h.
With a small drop of alcohol the eggs were transferred to a clean slide. As long
as the alcohol had not evaporated it remained possible to orient the eggs, usually
with the vegetative pole facing the slide. Evaporation of the alcohol fixed the
eggs firmly to the slide. Moreover, this procedure made the eggs shrink considerably, and this had the advantage of concentrating the Feulgen-positive
material of the nuclei. Eggs of one lot were mounted on the same slide. Finally,
the slides were dipped in a solution of 2 % agar at 60 °C and dried again. The
agar film held the eggs to the slide, especially at the moment of transfer from
hydrochloric acid to tap-water. Hydrolysis was performed in 5 N - H C I at 25 °C
for \- h. The eggs were washed for 30 min in stagnant tap-water and stained for
I h with SchifT's reagent, washed three times for 2-3 min in SO2-water and
finally in running tap-water for £h. As only the nuclei were stained it was
difficult to localize the eggs. Therefore, the position of the eggs was marked with
indian ink after passage through alcohol 96 % and before mounting. As soon as
the alcohol had evaporated the eggs became visible and their position could be
marked. After dehydration the slides were mounted in Caedax.
The absorption of light by individual nuclei was measured at 565 nm by
means of a Zeiss microscope-photometer, similar to that described by
MacGregor (1968). Either the method of Ris & Mirsky (1949) was used which in
the aperture of the phototube was adjusted so as to contain the whole nucleus,
or the method of Swift (1950) in which the area for measurement had a diameter
of about 60 % of that of the nucleus.
354
J. A. M. VAN DEN BIGGELAAR
Results
In this paper the 1-cell stage (first cleavage cycle, first cell cycle) of a developing egg of Lymnaea is defined as the period between the extrusion of the second
polar body and the beginning of the first cleavage, the 2-cell stage is the interval
between the beginnings of the first and second cleavages, the 4-cell stage is
represented by the interval between the beginnings of the second and third
cleavages (Fig. 1). At the third cleavage the first quartet of micrometers la-Id is
formed. Subsequent cleavages are no longer synchronous, macro- and micromeres having their own cleavage rhythms.
2nd maturation
division
1-cell stage
t
1st cleavage
2-cell stage
4-cell stage
Fig. 1. The first three cell cycles of the Lymnaea egg. Each cycle represents the
interval between the beginnings of two successive divisions.
In order to detect whether the egg capsules prevent the penetration of Tdr
into the eggs, they were incubated either with or without the capsule during the
1-, the 2- or the 4-cell stage. Only in the autoradiographs of eggs incubated
during the 4-cell stage could grains be observed over the nuclei. The number of
grains in the group of eggs incubated within the capsule did not differ from that
in the group of decapsulated eggs. Consequently it was decided to incubate the
eggs within the capsule.
An increase in the incorporation of labelled Tdr from the 4-cell stage was
substantiated by the results of eight experiments in which the reduplication time
was studied. For each experiment only one egg mass was used, which was
divided into synchronously dividing groups. The eggs were incubated at successive intervals during the 2- and the 4-cell stage. The duration of the corresponding stages within different egg masses was unequal. The average duration of the
2- and the 4-cell stage was 88 ± 8 and 86 + 8 min, respectively. To summarize the
results of the experiments, the actual incubation times have been transposed to
relative times by conversion to a cell cycle of the average duration (Table 1).
Mitotic stages observed at the end of the incubation periods are indicated in the
same table. It must be realized that it was difficult to establish the transition from
interphase to prophase and from telophase to interphase, because the staining
intensity of the nuclei with methyl-green-pyronine was very weak.
Timing of the phases of the cell cycle in Lymnaea. /
355
Table 1. Incorporation of[3H]Tdr at the 2- and the 4-cell stages
(The average duration of these stages was 88 ± 8 and 86 ± 8 min, respectively. The actual
incubation times have been transposed into relative times by conversion of the actual duration
of a cell cycle into a cycle of the average duration.)
Relative
incubation
time (min)
after the
onsel:of
Percentage of eggs in
t
N
2-cell
stage
4-cell
stage
6-26
10-37
18-37
31-51
39-56
40-62
47-64
39-67
49-69
56-74
59-80
48-88
69-88
—
—
—
—
—
—
—
—
—
—
—
—
—
-5
-6
-9
-9
-10
-11
466246786277—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
23
0-17
2-20
5-26
11-32
20-40
22-41
23-44
24-44
25-46
29-47
30-50
31-52
34-54
35-55
37-58
44-66
50-71
51-77
61-79
No.
of
Eggs
labelled
eggs
(%)
17
7
30
14
8
7
8
8
7
5
11
39
15
43
19
20
—
—
—
—
—
—
—
—
—
—
—
—
—
—
n
Interphase
Prophase
Prometaphase
100
100
100
79
—
—
—
—
—
—
—
—
—
—
—
5
—
26
12
100
—
—
—
21
100
86
12
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
10
83
62
71
—
—
—
—
—
—
—
—
—
14
63
50
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
29
33
—
—
—
31
8
4
35
33
39
38
100
2
100
100
13
42
5
14
20
16
13
24
12
30
100
100
100
57
60
25
62
—
—
—
—
—
—
—
—
—
—
100
100
100
100
100
100
100
100
100
90
17
38
—
—
—
—
—
6
30
16
7
6
6
5
6
>
Metaphase
Anaphase
Telophase
—
—
—
—
—
—
25
50
100
80
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
67
100
40
—
—
—
—
—
—
—
—
—
—
20
100
10
5
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
60
100
—
—
—
—
—
—
—
—
—
—
—
90
95
100
100
95
100
74
88
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
E M B
26
356
J. A. M. VAN DEN BIGGELAAR
From Table 1 it appears that in none of the eggs incubated during the 2-cell
stage could an incorporation of [3H]Tdr be detected. The first visible signs of
mitosis were observed 51 min after the beginning of the 2-cell stage. The second
cleavage started while the eggs were in late anaphase or early telophase. From 5
up to 11 min after the beginning of the 4-cell stage the eggs were in telophase; the
first positive autoradiographs were already obtained from eggs fixed 6 min after
the onset of cleavage. Thus a presynthetic or Gx phase must be absent. Subsequent groups with labelled nuclei could be obtained if the incubation started
earlier than 29 min after beginning of division. From sections extracted with
DNase prior to the application of the film, positive autoradiographs have not
been obtained. Sections treated with buffer solution were labelled to the same
extent as untreated controls. This proves that the incorporation of [3H]Tdr
indicates DNA synthesis. The most probable interval for the reduplication of
DNA during the 4-cell stage is between 5 and 29 min; that is, between 7 % and
34 % of this stage. A postsynthetic or G2 phase of about 23 min (27 %) must be
present as mitosis started at about 51 min.
Prior to the 4-cell stage no incorporation of [3H]Tdr was detected. However, a
very low rate of incorporation could not be excluded. Observation of a very low
rate of incorporation requires a much longer exposure time of the autoradiographs. For this reason, in the following experiments eggs were incubated at
different intervals during the 1- and the 2-cell stage. Experimental conditions
were the same as in the preceding experiments, but the average exposure time of
2 months was prolonged to 8 months. With this method positive autoradiographs
were obtained during both stages. As the eggs had not been synchronized the
reduplication time must be deduced in the following way. The average duration
of both the 1- and the 2-cell stage was 85 min. Assuming that the duration of
mitosis was the same as at subsequent stages, the eggs must have been in
prophase at about 50 min after the second maturation division and 50 min after
first cleavage, respectively. As in the preceding experiments, eggs fixed in
prophase were unlabelled. Since the incubation time was 20 min it can be
concluded that the reduplication must have been completed in the first 30 min
of both stages. Eggs with telophase nuclei fixed at the onset of the 2-cell stage
appeared to be labelled; as the incubation was started during the preceding
meta- or anaphase, this implies the absence of a Gx phase.
Timing of the phases of the cell cycle with Feulgen-stained nuclei
Introduction
The first attempts to demonstrate the presence of DNA in interphase nuclei of
eggs of Lymnaea during early cleavage stages by means of the Feulgen reaction
have been negative (Raven, 1946; Minganti, 1950). Negative results have been
reported also for eggs of sea urchins (Marshak & Marshak, 1953; Immers, 1957).
The proportionality between dye content and amount of DNA depends,
among other things, upon fixation and hydrolysis. Hydrolysis of DNA with
Timing of the phases of the cell cycle in Lymnaea. /
357
hydrochloric acid is an essential condition for the Feulgen reaction; it releases
purine bases and transforms DNA into apurinic acid. Depurination can be
performed in a wide range of temperatures. At high temperatures, however,
apurinic acid may be solubilized or extracted, thus reducing the amount of
Feulgen-positive material. If hydrolysis is performed at lower temperatures and
even if it is continued for several hours, the intensity of the Feulgen stain
remains undiminished (Jordanov, 1963; Decosse & Aiello, 1966; Bohm, 1968).
For this reason, it was decided to ^investigate the relationships in Lymnaea.
Hydrolysis was performed at 25 °C in 5 N-HC1. Nuclear staining was still
further intensified by shrinking the eggs, thus concentrating the Feulgen-positive
material.
Results
Timing of mitotic stages. It was difficult to synchronize the eggs before first
cleavage. The only reference point for synchronization was the extrusion of the
polar bodies and the duration of the mitotic cycle preceding first cleavage has
not been studied extensively. One series of observations has been performed.
After the extrusion of the second polar body the female chromosomes started
to swell into karyomeres. These chromosomal vesicles gradually coalesced and
formed the female pronucleus, which was irregular and usually ring-shaped.
Simultaneously, the sperm head was transformed into the regularly shaped and
homogeneously stained male pronucleus. The pronuclei became closely apposed
but did not fuse (Raven, 1945). Mitosis started 53 min after the extrusion of the
second polar body. First cleavage was reached after 90 min.
At the 2- and the 4-cell stage the mitotic cycle could be studied in detail. Eggs
were synchronized at the beginning of a cleavage, fixed at regular intervals,
Feulgen-stained and analysed for the duration of the different mitotic stages.
The cells were considered to be in prophase as soon as the first indication of a
linear organization of the chromosome material could be distinguished. At the
end of prophase the nuclear membrane dissappeared. Prometaphase represented
the movement of the chromosomes towards the equator of the spindle. During
metaphase the chromosomes were localized in the equatorial plane. At anaphase
the sister chromatids separated and moved towards the opposite poles of the
spindle. At the end of chromosome movement telophase is reached.
The results for the 2- and the 4-cell stage are shown in Figs. 2 and 3, respectively. The results of different experiments have been summarized in the same way
as mentioned before (Table 1). The duration of the 2- and the 4-cell stage was
84 + 6 and 82 + 8 min, respectively. In both stages mitosis started approximately
at 50 min (60 % of the cycle).
Timing of the period of DNA synthesis by means of cytophotometry of Feulgenstained nuclei. Because of the lack of an easily visible reference point, synchronization before first cleavage was difficult. Besides, the female pronuclei were too
irregular to be measured with the applied techniques. Swift & Kleinfeld (1953)
23-2
358
J. A. M. VAN DEN BIGGELAAR
reported the same difficulty for eggs of the grasshopper Melanoplus differentialis.
Finally, the polar bodies were usually situated in the light path of the pronuclei,
especially of the female pronucleus. For these reasons, time and duration of the
first S phase was partly determined in one experiment only. Up to 30 min after
the extrusion of the second polar body the male pronucleus could be measured
separately. The results (Fig. 4) demonstrate that Feulgen stain of the male
pronucleus did not increase during the first 12 min. It seems reasonable to assume
that DNA synthesis in the male pronucleus would have been completed about
37 min after the extrusion of the second polar body. If the same is applicable to
the female pronucleus, it may be concluded that a Gx phase has been very short
or even absent, and that the pronuclei must have passed a G2 phase from 37 to
56 min after completion of maturation.
2nd cleavage
100-
80-
%
60"
40-
20-
10
Telophase
Interphase
Prophase
Metaphase
Telophase
Prometaphase Anaphase
Fig. 2. Duration of mitotic stages during the 2-cell stage. At short intervals after the
onset of the 2-cell stage about 10 eggs were fixed. Abscissa: time scale; ordinate:
% of nuclei in telophase ( # ) , interphase ( x ) , prophase (O), prometaphase ( + ),
metaphase (D), anaphase (A)-
The pattern of DNA synthesis during the 2- and the 4-cell stage is shown in
Fig. 5 and Fig. 6, respectively. At early telophase the measured values were
smaller than the theoretically expected diploid amount. Discrepancies between
the real dye content and the measured absorption could be expected during
mitosis when the stain is not uniformly distributed (Swift & Kleinfeld, 1953). At
late telophase the chromosomes swell into karyomeres resulting in a more
homogeneous distribution of the Feulgen dye. In this way proportionality is
Timing of the phases of the cell cycle in Lymnaea. /
2nd cleavage
359
I 3rd cleavage
100- • —
80-
60-
40-
20-
10
Telophase
48
Interphase
62|66
76|80
Prophase
Metaphase Telophase
Prometaphase Anaphase
Fig. 3. Duration of mitotic stages during the 4-cell stage. At short intervals after the
onset of the 4-cell stage about 10 eggs were fixed. Abscissa: time scale; ordinate:
% of nuclei in telophase (#), interphase (x), prophase (O), prometaphase ( + ),
metaphase ( • ) , anaphase (A).
2x-
..
f
20
I s
Extrusion of
2nd polar bod/
30
40
50
G,
60
70
80
H
90
100 min
(
First
cleavage
Fig. 4. Reduplication of DNA in the male pronucleus. The amount of DNA is
expressed in multiples of the relative amount of DNA present in the sperm head, x
(a haploid nucleus). Means and standard errors of determinations of Feulgen-dye
content in one experiment. Each point represents 2-7 nuclei.
360
J. A. M. VAN DEN BIGGELAAR
restored. Consequently, at the earliest telophase stages it was not possible to
distinguish between swelling of the chromosomes and the beginning of DNA
synthesis. However, it seems reasonable to assume that reduplication started
2x
10
t "
20
S
30
|
40
G2
50
60
70
|
M
80
90
100 min
|
First
Second
cleavage
cleavage
Fig. 5. Reduplication of DNA at the 2-cell stage. Amount of DNA expressed in
multiples of the relative amount of DNA, x, present in a haploid nucleus (sperm
head). Means and standard errors of the means of five experiments. Each point
represents approximately 25 eggs.
2x -
10
20
30
40
Ga
50
60
70
80
90
100 min
M
Second
Third
cleavage
cleavage
Fig. 6. Reduplication of DNA at the 4-cell stage. Amount of DNA expressed in
multiples of the relative amount of DNA, x, present in a haploid nucleus (sperm
head). Means and standard errors of the means of five experiments. Each point
represents approximately 25 eggs.
Timing of the phases of the cell cycle in Lymnaea. /
361
6 min after the beginning of cleavage when the blastomeres were in mid-telophase.
At that moment the absorption reached the diploid value. Reduplication was
completed after 30 min. It may be concluded that at the 2- and the 4-cell stage
the blastomeres did not pass through a Gx phase. As mitosis started at about
48 min (Figs. 2 and 3) the nuclei passed through a G2 phase of 18 min. For the
4-cell stage these results are confirmed by the autoradiographic data listed in
Table 1.
DISCUSSION
The cell cycle of a differentiated cell can be divided into four phases: mitosis
(M phase), followed by a postmitotic-presynthetic phase (G1 phase), the period
in which DNA is reduplicated (S phase) and the interval between the completion
of DNA synthesis and the onset of mitosis (G2 phase) (Howard & Pelc, 1953;
Stevens, Daoust & Leblond, 1953). It is characteristic of embryonic cells that
they do not pass through a Gx phase. Reduplication of DNA starting at telophase
has been reported for eggs of sea urchins: Arbacia punctulata (Black, Baptist &
Piland, 1967), Paracentrotus lividus (Pasteels & Lison, 1951; Nemer, 1962),
Strongylocentrotus purpuratus (Hinegardner, Rao & Feldman, 1964; Brookbank,
1970), of the sand dollars Dendraster excentricus (Brookbank, 1970) and Echinarachnius parma (Young, Hendler & Karnofsky, 1969), of the phasmid
Clitunmus extradentus (Bergerard, 1955), of the brine shrimp Artemia salina
(Fautrez & Fautrez-Firlefyn, 1953) and for the 2-cell stage in the amphibian
Xenopus laevis (Graham & Morgan, 1966). Apparently, the same holds true for
Lymnaea as during the first three cleavage cycles a Gx phase could not be
demonstrated.
After the second maturation division the two pronuclei pass separately
through the first S phase. Synthesis of DNA before nuclear fusion has been
reported similarly for the frog Xenopus (Graham, 1966), the sea urchin Paracentrotus lividus (Anderson, 1969), the sand dollar Echinarachniusparma (Simmel
& Karnofsky, 1961), the cricket Melanoplus differentialis (Swift & Kleinfeld,
1953), the caddis-worm Sabellaria (Pasteels & Lison, 1951) and the rat and
mouse (Alfert, 1950; Dalcq & Pasteels, 1955; Sirlin & Edwards, 1959). In
Lymnaea fertilization takes place in the spermoviduct while the eggs are in the
first meiotic metaphase (Bretschneider, 1948). Maturation is completed about
300 min later. During this period the sperm head remains localized beneath the
cell surface. Only after the end of the maturation divisions does it move towards
the centre of the egg, start to swell and finally become associated with the female
pronucleus. Karyogamy does not take place (Raven, 1945). Completion of the
second maturation division apparently triggers migration and swelling of the
male pronucleus and the beginning of DNA synthesis.
The difference between the rate of incorporation of exogenous Tdr before and
after second cleavage needs further comment. Reduplication of DNA and
synthesis of associated histone are energy-requiring processes. For this reason
362
J. A. M. VAN DEN BIGGELAAR
several authors postulated a relationship between reduplication of nuclear
material and consumption of oxygen (Zeuthen, 1951; Comita & Whiteley, 1953;
Holter & Zeuthen, 1957; Whiteley & Baltzer, 1958). A relation between the
incorporation of Tdr into DNA and the consumption of oxygen has been
demonstrated for HeLa cells by Robbins & Morril (1969).
The uptake of oxygen by individual eggs of Lymnaea during the 2- and the
4-cell stage has been measured by Geilenkirchen by means of the Cartesian diver
method (1961). In both stages the onset of mitosis coincides with a peak in the
uptake of oxygen. However, only at the 4-cell stage a superimposed peak
coincides exactly with the incorporation of [3H]Tdr into DNA.
The coincidence of an increased respiratory rate and an increased incorporation of exogenous Tdr into DNA observed at the 4-cell stage might indicate a
relation between DNA synthesis and oxygen consumption. If a causal relationship exists between these two processes, the number of oxygen molecules
represented by the peak superimposed on the basic pattern of the respiratory rate
must be of the same order of magnitude as the number of oxygen molecules
necessary for the production of high-energy-rich ATP molecules associated with
the reduplication of DNA and the synthesis of nucleohistone. This amount can
only be partly calculated. The insertion of each nucleoside into a strand of DNA
requires three molecules of ATP. In deoxyribonucleohistone three or four amino
acids are present per nucleotide (Bloch, 1963). One peptide bond is formed at
the expense of three energy-rich phosphate bonds (Lehninger, 1965). The DNA
content of a sperm cell of Lymnaea is 2 x 10~12 g (R. T. Hinegardner, personal
communication; J. J. Haaijman, unpublished results). If it is assumed that the
average molecular weight of the nucleotides is 333, then 0-72 x 1010 nucleotides are
present in a diploid nucleus and three to four times as many amino acids.
Insertion of these nucleotides requires at least 2-16 x 1010 ATP molecules, the
formation of peptide bonds ultimately requires about 7-56 x 1010 ATP molecules.
Consequently, to insert the theoretical amount of nucleotides and amino acids
into deoxyribonucleohistone at the 4-cell stage the embryo requires approximately 38-64 x 1010 molecules of ATP. This corresponds to about 6xlO 10
molecules of oxygen, for the oxidation of one molecule of glucose with six
molecules of oxygen with an efficiency of 42 % yields 38 molecules of ATP
(Lehninger, 1965). It can further be calculated from Geilenkirchen's data (1961)
that the respiratory peak superimposed on the basic pattern of respiration
during DNA synthesis at the 4-cell stage represents approximately 38 x 1010
molecules of oxygen. So, if it is correct to relate this increased oxygen uptake
with reduplication of DNA, a number of other energy-requiring processes must
also be involved. One of these processes might be the activation of enzymes
associated with DNA synthesis. In Escherichia coli the phosphorylation of Tdr
into dTMP requires two molecules of ATP; one molecule as a phosphate donor
and one for the activation of Tdr kinase, i.e. to increase the affinity of the
enzyme for its substrate (Okazaki & Kornberg, 1964). Another energy-requiring
Timing of the phases of the cell cycle in Lymnaea. /
363
process might be the synthesis of template RNA for the production of nuclear
protein. During the S phase of HeLa cells a class of small polyribosomes appears
(Robbins & Borun, 1967). In sea-urchin eggs light polyribosomes seem to be
engaged in the production of histones (Kedes, Gross, Cognetti & Hunter, 1969;
Nemer & Lindsay, 1969). Therefore it is not unlikely that the nucleus produces
messenger RNA for the synthesis of histone. At the moment, however, there
is no experimental evidence that in Lymnaea histones as well as the required
templates are produced during the DNA synthetic phase.
During the S phase of the 2-cell stage no change in the basic pattern of
oxygen consumption has been observed (Geilenkirchen, 1961). During the same
stage [3H]Tdr is only sparingly incorporated into DNA. This might be explained
by the existence of a pool of phosphorylated precursors already present in the
unfertilized egg. The presence of a pool of acid-soluble deoxyribonucleotide
precursors in the unfertilized egg has been demonstrated in a number of
organisms (Grant, 1965). A supply of phosphorylated precursors will reduce the
amount of ATP molecules necessary for DNA synthesis, and concomitantly the
increase in the respiratory rate will be less pronounced or even absent. Another
possible explanation might be a difference in the permeability of the egg to the
label. However, in a separate series of experiments (to be published elsewhere),
no difference in the uptake could be observed.
Summarizing, it may be concluded that it is hard to substantiate or to refute
the theory that an increase in respiration during the S phase of the 4-cell stage is
connected with the energy requirements of DNA synthesis.
RESUME
Determination des phases du cycle cellulaire par la thymidine tritiee
et Feulgen cytophotometrie pendant la periode de division synchrone
chez Lymnaea
La duree de la mitose et de la reduplication de l'ADN a ete determined par l'incorporation
de la [3H]thymidine et par la determination photometrique de la teneur relative en ADN dans
les stades a 1, 2 et 4 cellules du developpement de l'ceuf de la lymnee.
Jusqu'au stade a 8 blastomeres la mitose prend 48 %, la reduplication de l'ADN 27 % et la
phase G2 25 % de la duree du cycle. Les cellules ne passent pas par une phase Gi.
L'incorporation de la thymidine exogene est largement augmentee au stade a 4 blastomeres,
ce qui coincide avec une elevation de la respiration pendant la reduplication de l'ADN. Au
stade a 2 cellules, quand l'incorporation est plus moderee, la respiration basale n'est pas
changee.
La supposition est discussiee que 1'elevation de la respiration pendant la phase S soit liee
avec la reduplication du desoxyribonucleohistone.
This paper was prepared from a part of a doctoral thesis. This work was supported by a
grant from the Netherlands Organization for the Advancement of Pure Research (Z. W. O.).
The cytophotometrie facilities of Professor Dr M. T. Jansen and Miss C. W. Leeflang are
gratefully acknowledged. The author wishes to thank Dr L. Boomgaart for improving the
English and H. A. Wagemaker for skilful assistance.
364
J. A. M. VAN DEN BIGGELAAR
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(Manuscript received 8 January 1971)