Differentiation 17, 111-115 (1980) Differentiation

0 Springcr-Verlag 19x0

Histone Analysis During the First Cell Cycle

of Development of the Sea Urchin Tetrapygzzs niger
MARIA IMSCHENETZKY, MARCIA PUCHI, and RINA MASSONE
Departamento de Bioquimica, Instituto de Ciencias MCdico Biolbgicas, Universidad de Concepci6n,
Casilla 2407, Concepci6n, Chile

To obtain information on the contribution of paternal and maternal histone complement to the
assembly of sea urchin chromatin after fertilization, we have compared the complete set of
histones of zygotes obtained at the beginning of the first S phase those of sperm and unfertilized
eggs of Tetrapygus niger. The electrophoretic pattern on acetic acid-urea polyacrylamide gels
and the amino acid composition of the histones isolated from zygotes are virtually identical with
those isolated from unfertilized eggs. These results strongly suggest that sperm histones must be
lost before the initiation of the first replication event. The histones of zygotes obtained after the
completion of the first S phase have the same electrophoretic pattern as the proteins isolated
from zygotes prior to the first S phase; however, differences were found in their amino acid
composition. These results suggest that some new proteins may associate with chromatin during
the first replication round in Tetrapygus niger development.

Introduction electrophoretic mobility may have different primary

Cleavage stages of sea urchin development are
characterized by very rapid DNA synthesis and cell
division. During this period a progressive increase in
the synthesis of early histone types takes place. These
early histones (a subtypes) are replaced at hatching
by two or more variants of each histone type (j3, y , 6
subtypes) [l-31.
Histone synthesis has been shown to begin at the
first cleavage division, and the newly synthesized
histones are associated with chromatin during the first
S phase [4-61. Therefore, it may be expected that by
the end of the first cell cycle, one half of the histone
complement of zygotic chromatin will be derived
from newly made histones, and from histones stored
in unfertilized eggs [7]. However, at the beginning of
the first S phase the histones present in chromatin will
be derived exclusively from the gametes.
It is well established that the histones of sea
urchin sperm differ from their counterparts in
unfertilized eggs, both in their electrophoretic mobil-
ity and in their amino acid composition [8-111. It is
also well known that histones with virtually the same
structures [12], and vice versa, histones with different
electrophoretic mobilities may have the same amino
acid sequence but differ in postsynthetic side chain
modifications [13]. Therefore, our approach has been
to compare the electrophoretic mobilities and the
amino acid composition of the complete set of
histones isolated from zygotes with those isolated
from sperm and unfertilized eggs. Here we present
evidence that before the first S phase zygotic
chromatin contains only histones of the maternal
type. Furthermore, our data strongly suggest that
some new proteins are assembled into chromatin
during the first replication event of Tetrapygus niger
zygotes.
Methods
Gametes and Embryos
Tetrapygus niger were collected in Concepci6n Bay, Chile. Sea
urchins were maintained in the laboratory in aquaria filled with sea
water for a period varying between one and six days. Female

0301-4681/80/0017/0111/$01.00
112 M. Imschenetzky et al.: Histones in First Cell Cycle of Sea Urchin Development
gametes were obtained by gently shaking gonads in sterile sea
water. Male gametes were collected after injection of 0.55 M KCI
into the coelomic cavity [14].
The handling of embryos was performed according to standard
methods. Eggs were filtered through a 100 pm pore size plancton
network and washed several times with sterile sea water.
Insemination of eggs was perfomed as described by Hinegardner
[15]. Development took place at 18C in sterile sea water with
constant aeration. Only embryos cultures with synchronization
over 95% were selected. A part of each culture was allowed to
develop upto pluteus stage, as a control.
Chromatin Purification and Histone Isolation
All procedures for the isolation of chromatin and histones were
carried out between 0-4" C. Unfertilized eggs or zygotes (60ml)
and 5 ml of sperm were washed several times with 1.5 M dextrose
and centrifuged each time at 750 g for 10 min (8,000g for 10 min in
the case of sperm). The resultant pellets were homogenized by five
to seven strokes of a Potter-Elvehjem homogenizer fitted with a
teflon pestle in 10 volumes of 50 mM EDTA buffer, pH 6.0
adjusted with NaOH. Under these experimental conditions
proteolysis of histones is not observed [16]. To separate the
unbroken cells. the homogenate was filtered through a plancton
net (45 pm pore size) and centrifuged at 4.000 g for 15 min. The
supernatant was discarded, and the pellet was resuspended in 10
volumes of 1 M sucrose, 50 mM EDTA buffer, pH 6.0, 0.2%
Triton X-100, and centrifuged at 12,000 g for 15 min. This
procedure was repeated twice. The chromatin pellet was conse-
cutively washed in 10 volumes of 50 mM EDTA buffer, pH 6.0;
5 mM EDTA buffer, pH 6.0; and 1 mM EDTA buffer, pH 6.0, and
centrifuged at 10.000 g for 15 min each time. The chromatin pellet
was subsequently washed in tridistilled water and centrifuged as
indicated above; this procedure was repeated until no appreciable
amount of pigment was observed in the pellet.
Histones were extracted from chromatin by stirring overnight
in 0.4 N H2S04 at 4" C. Acid-insoluble material was removed by
centrifugation at 30,000 g for 20 min and the supernatant
containing the histones was concentrated. After dialysis against
tridistilled water the material was lyophilized and stored at
-20C.
Polyacrylarnide Gel Electrophoresis
Histones isolated from unfertilized eggs, zygotes, and sperm were
dissolved in 6 M urea, 2% 0-mercaptoethanol, 0.9 N acetic acid.
Samples containing 50-150 pg of histones were run in 15 cm slab
gels containing 15% acrylamide, 0.2% bisacrylamide, 6 M urea,
0.9 N acetic acid. Gels were pre-electrophoresed for 4 h at 40
mAmp, and the histone samples were run under the same
conditions. Gels were stained in 1% Amido Black, 7% acetic acid,
and destained in 7% acetic acid, 40% methanol. Scanning was
performed on a Gilford spectrophotometer (Oberlin, Ohio)
equipped with a linear transport gel scanner.
Protein concentration was measured according to the method
of Lowry et al. [17]. In samples containing 6 M urea, protein
determinations were perfomed according to the method described
by Luck et al. [ 181. Calf thymus histones, purchased from Sigma
Chemical Co. (St. Louis, Missouri), were used as standards.
Amino Acid Analysis
The lyophilized protein was hydrolyzed at 100"C for 24 h in small
evacuated flasks containing 1 ml of 5.7 N HCI; amino acids were
determined with the standard column of an amino acid analyzer
DURRUM 500 D. (Palo Alto, California).
Tryptophane was determined by fluorescence measurement in
an Aminco-Bowman spectrophotofluorometer (Silver Spring,
Maryland). The fluorescenceof the protein solution was compared
at 345 nm with tryptophane, and bovine serum albumin was used as
standard. The excitation of fluorescence was performed by
monochromatic light at 280 nm [19].
Assay for DNA Synthesis
Unfertilized eggs were prelabeled for 90 min at 18" C with
3[H]-thymidineat a concentration of 7.5 pCdml (NEN, sp. act. 47.6
Ci/mole). At different times after fertilization samples were
removed, washed once with cold sea water, twice with 1.5 M
dextrose, and centrifuged each time at 750 g for 5 min. The pellet
was homogenized in 50 mM EDTA buffer, pH 6.0; 2% Triton
X-100 and centrifuged at 4,000 g for 15 min. The resulting nuclear
pellet was washed twice with 5 mM EDTA buffer pH 6.0, and then
suspended in 1N NaOH for determination of radioactivity and
protein concentration. 3[H]-thymidineincorporation into the acid
insoluble fraction was performed by the paper filter disc method as
described by Bollum [20]. Radioactivitywas measured in a Nuclear
Chicago ISOCAP 300 (Des Plaines, Illinois) scintillation counter
(counting efficiency 39%).
Results
D N A Synthesis During the First Cleavage Cycle
To determine cell cycle parameters during the first
cleavage division, unfertilized eggs were preloaded
with 3[H]-dlTP for 90 min prior to fertilization.
7 20
Time ( min )
Fig. 1. Incorporation of 3[H]-dTTPinto DNA of Tefrapygus niger
zygotes. The sperm suspension was added at zero time
M. Imschenetzky et al.: Histones in First Cell Cycle of Sea Urchin Development
Samples were removed at various times after insem-
ination and 3[H]-dTTPincorporation into the nuclear
acid-insoluble fraction was determined. As shown in
Fig. 1 the first S phase occurred between 40 and 90
min after fertilization.
ZYGOTE 90
ZYGOTE 40
113
Analysis of Histones
During the First Cleavage Cycle
The electrophoretic mobility and the amino acid
composition of histones from zygotes at the beginning
of the first S phase (40 min after fertilization) were
compared with those obtained from sperm and
unfertilized eggs. We also examined histones
obtained from zygotes that had completed the first
replication round (90 min after fertilization).
In Fig. 2 are shown the electrophoretic profiles of
histones from both gametes and zygotes before and
after the first replication event. In eggs and zygotes,
there were proteins that co-electrophorese with the
nucleosomal core histones H3, HZB, HZA, and H4.
Two closely migrating bands were detected in the H4
position, suggesting a postranslational modification
on this fraction. No band was seen in the exact
position of the sperm HI histone, but some other
bands were observed in the H1 region of the gel: a
doublet migrating between H1 and H3 and two other
bands with lower mobility than the sperm HI fraction.
In sperm, H3 was present at a higher proportion than
H2B and HZA,whereas in zygotes as well as in eggs this
fraction was a minor component of the H3-HzB-HZA
region.

UNFERTILIZED
EGG Table 1. Amino acid composition of histones isolated from
chromatin of Terrapygus niger (mol%). Tryptophane was deter-
mined by spectrofluorometry (181
Amino acid Amino acid composition of histones frac-
'tions

Egg Zygote Zygote Sperm

40 min 90 min
Lysine 10.91 10.99 9.59 14.82
Histidine 2.00 1.98 2.14 1.16
Arginine 6.81 7.05 6.10 11.81
Aspartic acid 11.71 11.59 10.45 5.16
Threonine 5.05 6.33 5.56 5.32
Serine 8.89 9.45 7.38 5.97
Glutamic acid 12.17 13.78 12.12 7.54
Proline 6.95 5.95 6.28
~~ 5.18
Glycine 13.68 12.64 9.55 8.80
I 'r, I
Alanine 11.01 10.95 8.83 14.94
H4 H2* H3 H1 4.08 4.79 5.09 4.55
Valine
"23 Methionine - - 1.85 1.01
Fig. 2. Comparison of the electrophoretic patterns of histones Isoleucine - - 3.59 3.25
isolated from gametes and zygotes of Tetrapygus niger: sperm Leucine 5.95 6.41 7.04 7.17
histones (50 pg) were run in the same gel with 100 pg of histones Tyrosine - - 1.85 1.55
from unfertilized eggs; 120 pg of histones from zygotes obtained 40 Phenylalanine - - 2.56 1.76
min after fertilization and 120 pg of histones from zygotes obtained Tryptophane none none none none
90min after fertilization. The direction of migration is from right to Lysine/Arginine 1.60 1.56 1.58 1.27
left, cathode to anode
114 M. Imschenetzky et al.: Histones in First Cell Cycle of Sea Urchin Development
Further evidence that the histones of zygotes
before the first S phase were of maternal origin was
provided by amino acid analysis. As shown in Table 1
the amino acid composition of whole histones isolated
from zygotes before the first S phase is virtually
identical to that obtained from unfertilized eggs.
Sperm histones contained more arginine, and the
amount of glutamic and aspartic acid residues was
significantly lower than in histones from unfertilized
eggs.
The electrophoretic patterns of histones from
zygotes before and after the first S phase were very
similar, but differences were found in their amino
acid compositions. The histones isolated from zygotes
40 min after fertilization had more alanine, glycine,
and serine. In these fractions methionine, isoleucine,
tyrosine, and phenylalanine were not detected.
Discussion
This study indicates that the histones bound to
chromatin of Tetrapygus niger zygotes at the begin-
ning of the first S phase are virtually identical with
those isolated from unfertilized eggs. At the stage of
development analyzed, sperm histones, if conserved,
should account for one half of the histones present in
zygotes. Thus, the amino acid composition of whole
histones isolated from zygotes should differ from
those obtained from unfertilized eggs. In particular, it
may be expected that the histones isolated from
zygotes contain a higher amount of arginine, lysine,
and alanine, which are the major amino acids of
sperm proteins, and also lower amounts of glutamic
and aspartic acid residues. However, the identity of
histones isolated from zygotes prior to S phase and
those isolated from unfertilized eggs strongly suggests
that sperm histones must be lost sometime before the
first replication event. Under the experimental
conditions used in the present work, histone pro-
teolysis is completely avoided [16], so loss of sperm
histones during isolation is very unlikely.
The analysis of histones bound to chromatin after
the first replication event indicates that they are
partially different from maternal fractions. In gen-
eral, however, their electrophoretic pattern is iden-
tical to that of maternal histones (Fig. 2 and Table l),
but, their amino acid composition reveals the pres-
ence of some residues that are not detected in
histones from eggs or from zygotes prior to the first S
phase. This difference strongly suggests that one or
more new proteins are bound to chromatin during the
first S phase. Histones having identical electropho-
retic mobilities but different amino acid composition
have been previously described in sea urchins [12,
131. The assembly of newly synthesized histones into
chromatin during the first cell cycle has been
observed in Arbacia punctulata [4, 51 and also in
Tetrapygus niger [6].
A problem in the isolation of histones from
unfertilized eggs as well as from early cleavage stage
embryos is the high nucleocytoplasmic volume ratio;
thus, the probability of contamination of histones
with cytoplasmic proteins is fairly high. However,
most cytoplasmic proteins, ribosomal proteins
included, do contain tryptophane. Histones do not
contain this amino acid, therefore, the amount of
tryptophane may be regarded as a criterion for
histone purity. Since none of the proteins analyzed
contains tryptophane in detectable amounts, contam-
ination with significant amounts of protein of cyto-
plasmic origin is very improbable. Furthermore, we
have previously shown that 3[H]-histones synthesized
de novo are not bound to chromatin of zygotes before
the first replication round [6], therefore a contami-
nation with cytoplasmic histones is unlikely. How-
ever, some non-histone chromosomal proteins may
be coextracted with histones from unfertilized eggs
and from zygotes, in particular high mobility group
(HMG) proteins, which also have a high content of
basic amino acids and are of low molecular weight
[211.
Our results are in agreement with those obtained
by Carroll and Ozaki [8], regarding the loss of sperm
histones during the first cleavage cycle of Strongy-
locentrotus purpuratus. However, at variance with
their work we find three major proteins in zygotes
and in unfertilized eggs with electrophoretic mobil-
ities similar to HI histone. These proteins do not
disappear when electrophoresis is perfomed on
sodium dodedecyl sulfate (SDS) gels, or on Triton
DF-12 gels (unpublished observation). The presence
of similar proteins migrating in the HI region of the
gel (cleavage stage fractions) has also been detected
in Strongylocentrotus purpuratus during the second
and third S phase [2]. The discrepancies on this point,
between our results and those of Carroll and Ozaki
may be due to differences in the procedures
employed for chromatin isolation, as HI histone or
related fractions are more suceptible to loss during
isolation than other histones.
Acknowledgements. The authors wish to express their gratitude to
Professor Jean Brachet who very kindly offered us the possibilityof
performing part of this work at the UniversitC Libre de Bruxelles
and for his very helpful criticisms, comments, and suggestions in
the preparation of the manuscript. We also want to thank Dr.
M. Imschenetzky et al.: Histones in First Cell Cycle of Sea Urchin Development
Catherine C. Allende from the Biochemistry Department Uni-
versidad de Chile and Drs. Janet Stein and Gary Stein from the
Department of Biochemistry and Molecular Biology of the
University of Florida U.S.A., for their very helpful suggestionsand
for correcting the English of the manuscript.
Amino acid composition determinations were performed in
the Laboratorium voor Chemie der Proteinen, Vrije Universiteit
Brussel and are hereby greatfully acknowledged.
This work was supported by grants from Programa RLA
76/006 PNUD UNESCO, from Programa Regional de Desarrollo
Cientifico y Tecnolbgico de O.E.A., and from V.R.I. Universidad
de Concepcibn.
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