3291
Journal of Cell Science 107, 3291-3300 (1994)
Printed in Great Britain © The Company of Biologists Limited 1994
Nuclear distribution of proliferating cell nuclear antigen (PCNA) in fertilized
eggs of the starfish Asterina pectinifera
Akira Nomura
Department of Zoology, Faculty of Science, Kyoto University, Kyoto 606, Japan
Present address: Tateyama Marine Laboratory, Ochanomizu University, Koh-yatsu, Umi-no-Hoshi, Tateyama, Chiba, 294-03, Japan
SUMMARY
Previous studies (Nomura et al. (1991) Dev. Biol. 143, 289296 (1993) Dev. Biol. 159, 288-297) determined the time of
DNA replication period (S phase) in starfish eggs fertilized
either during or after oocyte maturation. Here proliferating cell nuclear antigen (PCNA) localized within nuclei of
starfish eggs was detected with an anti-PCNA human
antiserum. Using a confocal laser scanning microscope, a
three-dimensional structure of the PCNA region was
analyzed.
In eggs fertilized during maturation, PCNA started to
localize within the nuclei at the same time as the initiation
of the first S phase. During the S phase, the distribution of
localized PCNA in a three-dimensional view coincided with
the chromatin distribution. After the S phase, PCNA
remained localized within the nuclei, but its distribution no
longer coincided with the chromatin distribution.
In eggs fertilized after maturation, however, PCNA
started to localize within the female pronuclei about 10
minutes ahead of the first S phase. Localized PCNA
occupied only a limited region of the nuclei without
diffusing over the whole nuclear area. Chromatin distributed around the peripheral region of the nuclei mostly
outside the PCNA region. When the first S phase was
initiated, the chromatin distribution became coincident
with the PCNA region. Later behavior of PCNA was the
same as that of the eggs fertilized during maturation. The
precocious localization of PCNA in those eggs fertilized
after maturation simply demonstrates that the ‘postactivation process’ for preparing DNA replication is triggered by
fertilization and PCNA localization and S phase are
sequentially initiated with a time-lapse. On the other hand,
the simultaneous occurrence of them seen in those eggs fertilized during maturation indicates that the postactivation
process must be going on in parallel with the maturation
process.
INTRODUCTION
before polar body formation will be called ‘early fertilized
eggs,’ and the eggs fertilized after the completion of the maturation process will be called ‘late fertilized eggs.’
Proliferating cell nuclear antigen (PCNA) has been a wellknown protein essential for DNA replication in many types of
cells (Tan et al., 1986; Bravo et al., 1987; Prelich et al., 1987;
see also Fairman, 1990, for review). Association of PCNA with
nuclei during the DNA replication period (S phase) was first
found in mouse NIH 3T3 cells fixed with methanol: PCNA was
detected in the nuclei only during S phase, and it was easily
extracted from the nuclei during phases other than S phase
(Bravo and Macdonald-Bravo, 1987). Extensive studies have
shown important roles for PCNA in DNA replication (Bravo,
1986; Nakamura et al., 1986; Fairman et al., 1988). PCNA was
finally identified as an auxiliary protein of DNA polymerase δ
(Bravo et al., 1987; Prelich et al., 1987). It is now certain that
PCNA binds to the replication fork, and is an indispensable
protein molecule for the DNA replication system (see van der
Vliet, 1989; Laskey et al., 1989, for review).
In 1989, Takasaki and his colleagues found that a human
antiserum from an auto immune patient recognized not only
human PCNA but also higher plant PCNA (Suzuka et al.,
Starfish oocytes resume their maturation by 1-methyladenine
(1-MA) treatment, and complete it without any arrest, followed
by formation of the female pronucleus (Fig. 1A). The absence
of ‘metaphase arrest’ is a characteristic feature of starfish
oocytes. After the germinal vesicle breakdown (GVBD), they
can be fertilized at any stage of their maturation (Fig. 1B,C).
Thus, both oocytes and eggs are fertilizable. Irrespective of the
time of fertilization, later development is apparently normal. If
the eggs are fertilized after the completion of maturation and
the formation of female pronuclei, DNA replication is initiated
30-50 minutes after fertilization (Nomura et al., 1991). This
indicates that the DNA replication system of starfish will be
established during this period of 30-50 minutes beginning from
fertilization. The process for preparing DNA replication was
called the ‘postactivation process’ by Nomura et al. (1991)
(Fig. 1C). In contrast, if eggs are fertilized before polar body
formation, DNA replication immediately follows pronucleus
formation (Nomura et al., 1991). This means that the postactivation process must be going on and completed in parallel with
the maturation process (Fig. 1B). From now on, eggs fertilized
Key words: starfish embryo, cell cycle, S phase, BrdU, PCNA,
CLSM, three-dimensional reconstruction
3292 A. Nomura
A
GVBD
PB1
PB2
Maturation process
B
Egg
GVBD
PB1
PB2
GVBD
Fertilization
pronuclei
1S
Postactivation process
2S
Egg
Maturation process
C
pronucleus
PB1
PB2
pronucleus
Postactivation process
1-MA
1S
Egg
Maturation process
1hr
2hr
Fertilization
1989). Dr Yoshinari Takasaki kindly gave this anti-PCNA
human antiserum for me to test whether it recognizes starfish
PCNA. In the previous study, I have already found a spatial
correlation between DNA replication sites and chromatin
structure (Nomura et al., 1993), and my present question is:
how does PCNA co-distribute with DNA replication sites or
chromatin? Methanol-fixed nuclei were exclusively observed
to study only those PCNA molecules incorporated into the
replication fork. The three-dimensional structure of the region
where PCNA and chromatin co-distribute was also observed.
MATERIALS AND METHODS
Cell culture and BrdU labeling
The starfish Asterina pectinifera were collected on the coast of
Wakasa Bay and Bohso Peninsula, Japan, and stored in aquaria in
Tateyama Marine Laboratory, Tateyama.
As described in a previous paper (Nomura et al., 1991), the oocytes
were: (i) induced to mature with 1 µM 1-methyladenine (1-MA); (ii)
inseminated; and (iii) timed visually for their developmental events.
Germinal vesicle break down (GVBD), and first and second polar
body (PB1 and PB2) formation occurred, respectively, about 20
minutes, 60-70 minutes and 90-100 minutes after 1-MA treatment at
20°C (cf. Figs 2, 3). They were inseminated at 40 minutes or 2 hours
after the 1-MA treatment. Eggs inseminated 40 minutes after the 1MA treatment are the ‘early fertilized eggs’ and eggs inseminated 2
hours after 1-MA are the ‘late fertilized eggs.’ Oocytes, eggs, and
embryos were cultured in a thermostatic bath at 20°C.
The fertilization membrane of the eggs was removed 5-10 minutes
after the insemination as described in a previous paper (Nomura et
al., 1993). Samples of eggs taken from the culture dish every 4
minutes were labeled with bromodeoxyuridine (BrdU; Sigma
Chemical Co., St Louis, MO) for 3 minutes in seawater containing 10
mM BrdU and 0.05% Triton X-100 (Sigma). After the 3 minute
labeling, they were fixed with methanol for 30 minutes at room temperature. They were then rinsed with phosphate-buffered saline (PBS)
at pH 7.3, three times. Some eggs in each sample were processed for
detection of the BrdU, and the other eggs were processed for staining
for PCNA.
Immunocytological procedures
BrdU incorporated into the nuclei of eggs was detected with an anti-
3hr
Fig. 1. Schematic illustration of temporal
correlation between the maturation process
and the postactivation process.
(A) Unfertilized eggs. Progression of the
maturation process is represented by the white
to gray gradation. The subsequent gray zone
indicates that the maturation process has been
completed. ‘Egg’ means a mature oocyte.
(B) Eggs fertilized during the maturation
process. The progression of the postactivation
process is illustrated by the inner rectangle
with the gradient of white to dark gray. Since
the postactivation process has been completed,
the first S phase (1S) is initiated when the
maturation process is completed. 2S, the
second S phase. (C) Eggs fertilized after
maturation. Since the maturation process has
been completed, 1S is immediately initiated
when the postactivation process is completed.
GBVD, germinal vesicle breakdown; PB1 and
2, first and second polar body formation.
BrdU monoclonal mouse antibody to time the length of S phase.
About 100 eggs were attached to a glass slide coated with poly-Llysine (Sigma). The eggs were incubated in a drop of a reagent or an
antibody in a moist chamber according to the following order: (i) 2
M HCl for 1 hour to denature the DNA; (ii) monoclonal mouse
antibody against BrdU (Becton Dickinson Immunocytometry
Systems, San Jose, CA) diluted to 1:6 with PBS for 2 hours; (iii)
biotinylated sheep monoclonal antibody against mouse Ig
(Amersham, Buckinghamshire, England) diluted to 1:50 with PBS for
1 hour; and (iv) FITC-conjugated streptavidin (Amersham) diluted to
1:100 with PBS for 1 hour.
PCNA was detected with an anti-PCNA human antiserum, and
DNA was counterstained with 4′-6-diamidino-2-phenylindole (DAPI;
Sigma), or propidium iodide (PI; Sigma). The eggs, attached to a glass
slide, were incubated according to the following order: (i) human
antiserum against PCNA (a gift from Dr Yoshinari Takasaki; Suzuka
et al., 1989) diluted to 1:100 with PBS for 2 hours; (ii) biotinylated
sheep monoclonal antibody against mouse Ig (Amersham, Buckinghamshire, England) diluted to 1:50 with PBS for 1 hour; (iii) FITCconjugated streptavidin (Amersham) diluted to 1:100 with PBS for 1
hour; and (iv) 0.2 µg/ml DAPI in PBS or 25 mg/ml PI in PBS for 1
hour. To use PI, eggs were preincubated in 2 mg/ml RNase A (Sigma)
at 37°C to digest the endogenous RNA that is stainable with PI. When
PBS was used for antibody dilution, it routinely contained 1% bovine
serum albumin (Sigma).
In western blot experiments, a 36×103 Mr band, consistent with
human PCNA (Mathews et al., 1984), was detected by the antiserum
against PCNA. Here the molecules recognized by this antiserum will
be tentatively regarded as ‘starfish PCNA.’
Finally, a drop of glycerol containing 10% 10mM Tris-HCl
adjusted to pH 8.0 and 2.3% 1,4-diazabicyclo[2.2.2.]octane (DABCO;
Sigma) was added to the eggs as an anti-fluorescence bleaching agent
and covered with a coverslip. The specimens were examined using
either a fluorescence microscope (Nikon Optiphoto with EFD2,
Tokyo, Japan) or a confocal laser scanning microscope (CLSM;
MRC600, Bio-Rad, Tokyo, Japan) equipped to an Axioplan epifluorescence microscope (Carl Zeiss, Germany).
Processing the images obtained by conventional
fluorescence microscopy
Images obtained by conventional fluorescence microscopy were
recorded on photographic film and were digitized by a filmscanner
(LS-3510AF, Nikon, Tokyo, Japan), and stored in a storage device of
Nuclear distribution of PCNA in starfish eggs 3293
A
Eggs (%)
100
GVBD
PB1
PB2
PCNA
CL1
CL2
CL3
CL4
50
0
2 BrdU
1
Fertilization
3
4
5
Time after 1-MA treatment (hr)
B
Eggs (%)
100
GVBD
PB1
PB2
PCNA
CL1
CL2
CL3
CL4
50
0
1
2 BrdU
3
4
5
Time after 1-MA treatment (hr)
Fertilization
a personal computer (Macintosh Quadra 950, Apple Computer Inc.,
Tokyo, Japan). Several kinds of digital image processing, such as
noise reduction and enhancement of the contrast, were made on the
original images with either an application software (Photoshop 2.5,
Adobe Systems Inc., Mountain View, CA) for digitally processing
photographic images or a specific filtering software (Kai’s power tool
2.0, Harvard Systems Corp., Santa Monica, CA).
Analyzing the images obtained with CLSM
Optical sections of stained PCNA and chromatin were obtained with
CLSM serially at a regular step of 0.48 µm. Each image was digitized
to numerical values (0-255) of 16×16 pixels for an area of 1 µm ×
1 µm on an optical section. The digitized images were stored in a
storage device of a computer (AX/2, Nimbus, Research Machines
Ltd, Oxford, UK) attached to the CLSM. The numerical data
were exported to a Macintosh Quadra 950 using a data-conversion
utility software (Access PC 2.1, Insignia Solutions Inc.). Several
kinds of digital image processing were made on the digitized images
with either a digital built-in filter within Photoshop or Kai’s power
tool.
The digitized images of the serial section at 0.48 µm step are too
far apart for three-dimensional reconstruction. Seven interpolated
images were then computed for each pair of neighboring images, and
inserted between them. This interpolation was done with an application software (Morph 2.0, Gryphon Software Corp., San Diego, CA)
for transforming digital graphic images. As a result, a single pixel
comes to represent the brightness of a cube of 1/16 µm × 1/16 µm
× 0.48/8 µm. All the digitized images including the interpolated
images were integrated into a three-dimensional image with an application software (VoxelView/Mac 1.0, Vital Images, Inc., Fairfield,
IA).
Fig. 2. Time course of early four cell cycles
showing a period of PCNA localization within
pronuclei at the first S phase in the early
fertilized eggs (A) and in the late fertilized eggs
(B). Solid circles and curves show the
percentages of PCNA-positive eggs. Gray
circles and curves are the percentages of eggs
labeled with BrdU. Crosses and broken curves
show germinal vesicle breakdown (GVBD), first
and second polar body formation (PB1 and PB2)
and the first through fourth cleavages (CL1CL4).
RESULTS
Timing the nuclear localization of PCNA
Starfish oocytes or eggs were fertilized either 40 minutes (early
fertilized eggs) or 2 hours (late fertilized eggs) after 1-MA
treatment. Their developmental events such as GVBD, polar
body formation, and cleavages were determined visually. BrdU
incorporated into the nuclei was detected with the anti-BrdU
antibody. In this study, time of S phase is routinely defined as
the time when more than 50% of eggs are labeled with BrdU.
PCNA localized within the nuclei was also detected with the
anti-PCNA antiserum. The percentages of PCNA-positive cells
and BrdU-positive cells are shown in Figs 2 and 3.
In the early fertilized eggs, both the nuclear localization of
PCNA and the first S phase were simultaneously initiated (Fig.
2A). In the late fertilized eggs, however, PCNA started to
localize about 10 minutes before the initiation of the first S
phase (Fig. 2B). The marked contrast in the time of PCNA
localization between the two types of eggs will be referred to
again. PCNA remained localized within the nuclei for 10-15
minutes after the end of the first S phase.
Localization of PCNA during the second and the third cell
cycle was also examined (Fig. 3). In the early fertilized eggs,
the behavior of PCNA was the same in each cell cycle as far as
examined: it started to localize at the same time as the initiation
of S phase, remained localized within the nuclei, and disappeared before the start of each metaphase (Fig. 3A). In the late
fertilized eggs, PCNA had already localized within the nuclei
3294 A. Nomura
A
Eggs (%)
100
GVBD
PB1
PB2 PCNA m1a1 CL1 m2 a2 CL2 m3 a3 CL3
CL4
50
0
2 BrdU
1
Fertilization
3
4
5
Time after 1-MA treatment (hr)
B
Eggs (%)
100
GVBD
PB1
PB2
m1a1 CL1 m2 a2 CL2 m3 a3 CL3
PCNA
50
2 BrdU
1
0
3
4
5
Time after 1-MA treatment (hr)
Fertilization
C
Eggs (%)
100
GVBD
PB1
PB2
PCNA
50
0
1
2
3
4
5
Time after 1-MA treatment (hr)
when the first S phase was initiated. In the second and the third
cell cycle, the behavior of PCNA was the same as in the early
fertilized eggs: it started to localize at the same time as the
initiation of S phase, remained localized within the nuclei, and
disappeared before the start of each metaphase (Fig. 3B).
If not fertilized, formed pronuclei remain quiescent for hours
without visible change. PCNA usually does not become
localized to such pronuclei. Occasionally, however, I found
some batches whose eggs were stained with anti-PCNA
antiserum at the pronucleus. The batch shown in Fig. 3 is an
example. Yet the time of the localization was widely scattered
among eggs (Fig. 3C), and I have no explanation of such an
unusual PCNA localization.
Fig. 3. Time course of early cell cycles showing
periods of PCNA localization within nuclei and S
phases in early fertilized eggs (A), in late fertilized
eggs (B), and in unfertilized eggs (C). Open
circles and thin curves are first through third
metaphases (m1-m3) and anaphases (a1-a3). Other
symbols and curves are the same as in Fig. 2.
The distribution of localized PCNA and the
chromatin structure
PCNA localized within nuclei was detected with the antiPCNA antiserum and stained with FITC. Nuclear DNA was
counterstained with DAPI. Fig. 4 illustrates a set of pictures of
eggs, derived from the batch shown in Fig. 2, taken with a conventional fluorescence microscope. The first S phases are
indicated by red frames surrounding the picture.
The results from the early fertilized eggs are shown in
columns A (chromatin structure) and B (localized PCNA).
Before the first S phase (one hour and 44 minutes after 1-MA
treatment, cf. Fig. 2A), both egg and sperm nuclei had formed
several small regions, indicating the formation of karyomeres
Nuclear distribution of PCNA in starfish eggs 3295
Fig. 4. Localized PCNA within nuclei in the fertilized eggs of starfish. PCNA was detected by the anti-PCNA antiserum, and stained with FITC
(columns B, D). DNA was also stained with DAPI (columns A, C). A series of images obtained from the early fertilized eggs are shown in
columns A and B, and those obtained from the late fertilized eggs are shown in columns C and D. These two kinds of series were obtained from
the same batch of eggs shown in Fig. 2. The numerals at the lower left corner of columns A and C indicate the time of fixation measured from
1-MA treatment (hour:minutes, for example 1:44 means the image obtained from eggs fixed at 1 hour and 44 minutes after 1-MA treatment). f,
female chromatin; m, male chromatin. Bar, 10 µm. The red frames surrounding the pictures at 1:56 and 2:28 indicate S phase as detected by
BrdU label (cf. Fig. 2).
3296 A. Nomura
Fig. 5. Reconstructed threedimensional images of the PCNA
region and the chromatin structure of
male and female pronuclei. This
image was made from the batch
shown in Fig. 2. The early fertilized
eggs were fixed at 1 hour and 56
minutes after 1-MA treatment
corresponding to the time when most
eggs are PCNA positive and BrdU
positive (cf. Fig. 2A). Localized
PCNA was stained with FITC
(green), and DNA was stained with PI
(red). Since two images were merged
into one, the yellow area represents
the co-distribution of both PCNA and
chromatin. (A) Outermost surface of
the reconstructed structure. (A1-A4) Four partitioned images made from the structure (A) to illustrate the inner part of the structure. Each
image was reconstructed from optical serial sections collected with CLSM, using the application software on the computer. Bar, 10 µm.
(Fig. 4, 1:44A). PCNA was not yet localized either in female
or male karyomeres (Fig. 4, 1:44B). During the first S phase,
localized PCNA was detected in both female and male
pronuclei (Fig. 4, 1:56). The distribution of localized PCNA
appeared similar to that of chromatin. After the end of the S
phase, PCNA remained localized, with the distribution nearly
coincident with that of chromatin (Fig. 4, 2:04). When
chromatin became condensed, PCNA was still detected but the
distribution was limited within a portion of nuclei (Fig. 4,
2:12). When chromatin condensed further, localized PCNA
was not detected (Fig. 4, 2:16). I have not clearly quantified
the correlation between a condensation level of chromatin and
the localization of PCNA.
The results from the late fertilized eggs are shown in column
C (chromatin) and D (PCNA). In this type of eggs, as was previously reported, female pronuclei had been formed at fertilization (Nomura et al., 1991). Male pronuclei derived from the
incorporated sperm nuclei were much smaller than the female
pronuclei (Fig. 4, 2:12C). Soon after fertilization, localized
PCNA was not yet detected either in the female or the male
pronuclei (Fig. 4, 2:12 D). As already shown in Fig. 2B, about
10 minutes before the initiation of the first S phase, PCNA
began to localize within the female pronuclei (Fig. 4, 2:24D).
It should be noted that the distribution of localized PCNA was
not coincident with chromatin that mainly distributed along the
peripheral region of the nuclei (Fig. 4, 2:24C). The nuclear
structure will be three-dimensionally examined later. During
the first S phase (red frames), localized PCNA was detected in
both female and male pronuclei (Fig. 4, 2:28D), and its distribution was coincident with chromatin (Fig. 4, 2:28C). After the
end of the S phase, PCNA remained localized, with the distribution nearly coincident with that of chromatin. Even within
condensing nuclei (Fig. 4, 2:56), PCNA was still detected, but
the distribution of PCNA was limited within a portion of the
nuclei. When chromatin condensed further, localized PCNA
was not detected (Fig. 4, 3:08).
In both early and late fertilized eggs, during the second cell
cycle, the distribution of localized PCNA was the same as that
of the first cell cycle in the early fertilized eggs.
Three-dimensional images of the PCNA region and
the chromatin structure
Using a computer graphics program, a structure where
localized PCNA was distributed was three-dimensionally
imaged on the computer display. Localized PCNA was
detected with the anti-PCNA antiserum and stained with FITC.
Nuclear DNA was counterstained with PI instead of DAPI,
since the CLSM used in this study did not detect the DAPIsignal. In my illustration of the reconstructed structure, PCNA
was shown in green color, and the chromatin was shown in red.
At first, images of the whole nuclei were reconstructed by
simply stacking the optical serial sections, and the outermost
surfaces of the nuclei were illustrated. To visualize the inner
part of the nuclear structure, two kinds of modifications were
made. One is dividing the reconstructed structure into several
partitions along an axis. They were illustrated as they were
separated from each other. These types of image will be called
‘partitioned images.’ The other modification is to make transparent the green area of localized PCNA. By this modification,
even the chromatin structure lying behind the green area
became partially visible through it. These types of image will
be called ‘transparent images.’ Images of both PCNA region
(green) and chromatin structure (red) were merged. Thus, the
area of chromatin where PCNA was co-distributed appears
yellow. Figs 5-9 show the results obtained from the batch
shown in Fig. 2.
A reconstructed image of the male and female pronuclei
during the first S phase in the early fertilized eggs is shown in
Fig. 5A, and its four partitioned images are shown in A1-A4.
At this stage, the distribution of localized PCNA extended to
the whole pronuclei, and entirely coincided with the chromatin
distribution, as shown by a predominant yellow area in the partitioned images (Fig. 5, A1-A4). After the end of the first S
phase, localized PCNA was distributed only within a certain
portion of the nuclei (Fig. 6A,B). Most PCNA was not co-distributed with the chromatin. Some chromatin assumed a
vesicular appearance, probably representing forming chromosomes. Some of them were wrapped around a zone of the
PCNA region (Fig. 6, B1-B4). This is well illustrated by the
mosaic pattern of the green, red, and yellow areas in the partitioned images (Fig. 6, A1-A4).
Nomura et al. (1993) reported that the chromatin of the
female pronuclei in the late fertilized eggs showed a hollow
structure. This hollow structure was also observed in the late
fertilized eggs examined in this study. Chromatin was distrib-
Nuclear distribution of PCNA in starfish eggs 3297
Fig. 6. Reconstructed three-dimensional
images of the PCNA region and the chromatin
structure of a nucleus. The early fertilized eggs
were fixed at 2 hours and 12 minutes when
most eggs are PCNA positive but BrdU
negative (cf. Fig. 2A). (A) Outermost surface.
(A1-A4) Four partitioned images made from
the structure (A). (B) Transparent image made
from (A). The green area of (A) was made
transparent. (B1-B4) Four partitioned images
made from the structure (B). Other
explanations are as for Fig. 5.
Fig. 7. Reconstructed three-dimensional
images of the PCNA region and the
chromatin structure of a female pronucleus.
The late fertilized eggs were fixed at 2 hours
and 24 minutes when most eggs are PCNA
positive but BrdU negative (cf. Fig. 2B). (A)
Outermost surface. (A1-A4) Four partitioned
images made from the structure (A). (B)
Transparent image made from (A). The green
area of (A) was made transparent. (B1-B4)
Four partitioned images made from the
structure (B). Other explanations are as for
Fig. 5.
uted mainly at the periphery and was sparsely distributed at the
central region of the nuclei (Fig. 7, A1-A4).
Before the first S phase (Fig. 7), localized PCNA (green)
clustered on one side of the inner space of the nuclei without
diffusing over the whole nuclear area. A part of the chromatin
was found within the PCNA region (Fig. 7, B1-B4). This is
well illustrated by the mosaic pattern of the partitioned images
(Fig. 7, A1-A4). When the first S phase was initiated (Fig. 8),
the mosaic pattern disappeared and the yellow area extended
widely in the partitioned images (Fig. 8, A1-A4). After the end
of the first S phase, the three-dimensional structure of the
nuclei was quite similar to that of the same stage in the early
fertilized eggs shown in Fig. 6. Localized PCNA was distributed only within a certain portion of the nuclei (Fig. 9A,B).
Most PCNA was not co-distributed with the chromatin. Some
vesicular chromatin was wrapped around a zone of the PCNA
region (Fig. 9, B1-B4). This is well illustrated by the mosaic
pattern in the partitioned images (Fig. 9, A1-A4).
Going back to Figs 7 and 8, I notice that the clustering of
PCNA distribution (green + yellow) on one side of the nuclei
seen before the first S phase (Fig. 7) persists through the S
phase (Fig. 8). This persistence of the clustering PCNA will be
referred to in the Discussion. On the other hand, the chromatin
(red + yellow) distributes around the peripheral area of the
nuclei mostly outside the PCNA region before the S phase (Fig.
7), and then becomes coincident with the PCNA region with
the initiation of the S phase (Fig. 8).
DISCUSSION
Use of the anti-PCNA human antibody for detecting
the nuclear localization of starfish PCNA
To detect a starfish equivalent of PCNA, the human anti-PCNA
antiserum was used. This antiserum is known to recognize
PCNA of various species, from higher plant PCNA to mammal
3298 A. Nomura
Fig. 8. Reconstructed three-dimensional
images of the PCNA region and the chromatin
structure of a female pronucleus. The late
fertilized eggs were fixed at 2 hours and 28
minutes when most eggs are PCNA positive
and BrdU positive (cf. Fig. 2B).
(A) Outermost surface. (A1-A4) Four
partitioned images made from the structure
(A). (B) Transparent image made from (A).
The green area of (A) was made transparent.
(B1-B4) Four partitioned images made from
the structure (B). Other explanations are as for
Fig. 5.
Fig. 9. Reconstructed three-dimensional
images of the PCNA region and the chromatin
structure of a nucleus. The late fertilized eggs
were fixed at 2 hours and 56 minutes when
most eggs are PCNA positive but BrdU
negative (cf. Fig. 2B). (A) Outermost surface.
(A1-A4) Four partitioned images made from
the structure (A). (B) Transparent image made
from (A). The green area of (A) was made
transparent. (B1-B4) Four partitioned images
made from the structure (B). Other
explanations are as for Fig. 5.
PCNA (Suzuka et al., 1989). In the present study, the molecule
in starfish eggs recognized by this antiserum was tentatively
called ‘starfish PCNA,’ on the basis of western blot analysis.
I found that the molecules recognized by the antiserum become
localized to chromatin in S phase nuclei, as do genuine human
PCNA molecules. This strongly indicates that the molecules
have such a function in DNA replication as to be properly
called ‘starfish PCNA.’
In DNA replication, PCNA molecules firmly bind to the
replication fork and are not easily extractable (see van der
Vliet, 1989; Laskey et al., 1989, for review). Based on results
obtained by Bravo and McDonald-Bravo (1987), localized
PCNA detected in this study is considered to represent those
that tightly bind to nuclei and are not extracted from nuclei
with the methanol fixation.
It should be a very interesting subject to relate the distribution of localized PCNA with that of DNA replication sites, by
simultaneous detection of PCNA and BrdU. Working preliminarily on starfish eggs, I realized, however, that the signal of
localized PCNA is often weakened after HCl treatment, a
procedure essential for the anti-BrdU antibody to recognize
incorporated BrdU. In a foregoing paper (Nomura et al., 1993),
I have already confirmed that the distribution of DNA replication sites detected by a BrdU pulse labeling was identical to
the chromatin distribution during the first and second S phases
of starfish eggs. Based on this observation, the chromatin distribution was taken to represent the sites of DNA replication
in this paper. Efforts were recently made to overcome the difficulty in simultaneously detecting DNA replication sites and
PCNA: Kill et al. (1991) used biotin-11-dUTP to label the
DNA replication sites, and Humbert et al. (1992) adopted an
enzymic procedure for denaturing the DNA instead of
chemical denaturation. I have not yet established if these
methods apply to starfish eggs.
Nuclear distribution of PCNA in starfish eggs 3299
Temporal correlation between the nuclear
localization of PCNA and the first S phase
A delay between the nuclear localization of PCNA and the
initiation of DNA replication has been noted (Celis and Celis,
1985; Hutchison and Kill, 1989; Nakane et al., 1989). The
technics improved by Kill et al. (1991) and Humbert et al.
(1992) have led them to confirm the presence of such a delay.
Kill et al. (1991) observed the delay in human diploid fibroblasts cells and Xenopus eggs, and proposed the idea that the
entry into S phase is biphasic: assembly of the replication
complex including PCNA is followed by a process in which
those complexes are used. Humbert et al. (1992) also observed
the delay and ascribed it to a time-lapse between the assembly
of replication complexes and the initiation of DNA replication.
Most recently, Baptist et al. (1993) proposed an unknown
process ‘X,’ programed after the nuclear localization of PCNA,
with a function necessary for DNA replication.
As I found in the late fertilized eggs of starfish, the precocious localization of PCNA within female pronuclei about 10
minutes before the initiation of the first S phase appears to generalize those delays mentioned above. It should be noted,
however, that the occurrence of the delay depends on the time
of fertilization in starfish: in the early fertilized eggs, PCNA
started to localize at the same time as the initiation of the first
S phase. What is unique in starfish eggs is that the time of fertilization alone makes such a contrast in PCNA localization.
Nomura et al. (1991, 1993) proposed the term ‘postactivation process’ to imply a cascade preparing for DNA replication in starfish eggs. The delay observed in the late fertilized
eggs indicates that the postactivation process induces the
nuclear localization of PCNA at 20 minutes after fertilization
and DNA replication at 30 minutes. Thus, localized PCNA
turns out to be a marker midway in the progress of the postactivation process. In the early fertilized eggs, both nuclear localization of PCNA and initiation of the first S phase immediately
followed the completion of the maturation process with the
formation of male and female pronuclei. These results support
the idea that the postactivation process must be going on and
must complete in parallel with the maturation process.
Spatial correlation between the distribution of PCNA
and the chromatin structure
In addition to the delay between the nuclear localization of
PCNA and the initiation of DNA replication, Kill et al. (1991)
observed that the preceding distribution pattern of PCNA often
resembles the expected distribution pattern of DNA replication
sites. Humbert et al. (1992) reported that a few of the PCNApositive/BrdU-negative cells show a PCNA distribution very
similar to the distribution at an early S phase.
Present studies on the late fertilized eggs of starfish
confirmed such a similarity, or a persistence of the PCNA distribution as a cluster on one side of the nucleus before and
during S phase. By merging PCNA and DNA label in the threedimensionally reconstructed images on the computer display,
I further observed that the precocious distribution of PCNA is
not identical to the chromatin distribution. This implies that
most PCNA molecules precociously localized within female
pronuclei cannot contact with the chromatin before the first S
phase. When the S phase is initiated, the distribution of PCNA
and chromatin become coincident with each other.
The persistence of the PCNA cluster before and during the
first S phase in turn will imply a dynamic relocation of the
chromatin mass towards the PCNA cluster for the onset of
DNA replication. Thus, S phase is correlated with a redistribution of chromatin to the PCNA sites in the nucleus. It appears
that such a relocation of chromatin mass has been unnoticed
heretofore, but the three-dimensional image analysis made in
the present study shows that it is actually the case at the start
of the first S phase at least in the late fertilized eggs of starfish.
If PCNA distribution can be looked upon as sites of functional
replication complexes, then my observation suggests that replication complexes are formed not strictly at replication forks
but as discrete sites in the nucleus that then become future sites
of replication.
In the early fertilized eggs and the second cell cycle of the
late fertilized eggs, the precocious localization of PCNA was
absent, and the distribution of PCNA was coincident with the
chromatin distribution at the start of the localization. This
suggests that PCNA properly reaches the DNA replication fork
as soon as the nuclei has been formed. The direct localization
of PCNA into the DNA replication fork should result in the
rapid start of DNA replication, which will help to shorten the
cell cycle periods in early development.
A built-in stability of the cell-cycle in starfish
embryos
No matter how the S phase initiation has a delay behind the
nuclear localization of PCNA, the disappearance of PCNA was
always scheduled at the initiation of chromatin condensation
(cf. Fig. 3). The second and the third cell cycle was quite
regular irrespective of the time of fertilization. In a previous
paper, Nomura et al. (1993) suggested that ‘the cell cycle
driving mechanism that is perturbed by the interaction of the
maturational process with the postactivation process could be
stabilized when the first round of the cell cycle is completed’.
Timely disappearance of localized PCNA from nuclei of the
starfish eggs as observed in the present study provides further
evidence for the operation of a certain device to ultimately
stabilize the cell cycle driving mechanism. As discussed earlier
(Nomura et al., 1993), this built-in stability of the cell-cycle
driving mechanism will be an essential device for normal
development of starfish eggs that are naturally fertilizable at
any time of maturation without the ‘metaphase arrest’ known
in most animal eggs.
I thank Dr Mitsuki Yoneda for reading the manuscript, and giving
helpful discussions. The western blotting experiment was done by Mr
Tatsuya Ueno of Kyoto University. I also thank Dr Yoshinari
Takasaki in Juntendo University for giving me the anti PCNAantiserum. I express my gratitude to Dr Shoji Tanaka in Mitsubishi
Kasei Institute of Life Sciences for instructing me to use the CLSM
at the institute and encouraging me to complete this work. Most of
the work was performed at Tateyama Marine Laboratory, Ochanomizu University, Umi-no-Hoshi, Tateyama, Chiba. I am grateful to
Dr Sin-ichi Nemoto, the director and Mr Mamoru Yamaguchi and Mrs
Hisae Kikuchi, staff of the laboratory for providing me with the facilities for research. I also thank Mr Tamio Arano in the ‘Zero-One
Shop, Cannon (Esaka branch)’ for his technical support to my
personal computer. I am grateful to Mr Toshimi Miyata, Mr Shin-ichi
Kajiya and other members in Risuken for giving me an opportunity
to master the computer programing. This work was supported in part
by a grant-in-aid for JSPS fellows.
3300 A. Nomura
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(Received 6 June 1994 - Accepted 16 August 1994)