Download Linker histone H1 in early mouse embryogenesis

2897
Journal of Cell Science 113, 2897-2907 (2000)
Printed in Great Britain © The Company of Biologists Limited 2000
JCS1314
Somatic linker histone H1 is present throughout mouse embryogenesis and
is not replaced by variant H1°
Pierre G. Adenot1,*, Evelyne Campion1, Edith Legouy1, C. David Allis2, Stefan Dimitrov3, Jean-Paul Renard1
and Eric M. Thompson1,4
1Unité de Biologie du Développement, Institut National de la Recherche Agronomique, F-78352 Jouy-en-Josas, France
2Department of Biochemistry and Molecular Genetics, University of Virginia Health Science Center, Charlottesville, Virginia
22908,
USA
3Laboratoire de Biologie Moléculaire et Cellulaire de la Différenciation, INSERM U 309, Institut Albert Bonniot, Domaine de la
Merci, 38706 La Tronche, Cedex, France
4Sars International Center, Bergen High Technology Center, Thormøhlensgt. 55, N-5008 Bergen, Norway
*Author for correspondence (e-mail: [email protected])
Accepted 9 June; published on WWW 20 July 2000
SUMMARY
A striking feature of early embryogenesis in a number of
organisms is the use of embryonic linker histones or high
mobility group proteins in place of somatic histone H1. The
transition in chromatin composition towards somatic H1
appears to be correlated with a major increase in
transcription at the activation of the zygotic genome.
Previous studies have supported the idea that the mouse
embryo essentially follows this pattern, with the significant
difference that the substitute linker histone might be the
differentiation variant H1°, rather than an embryonic
variant. We show that histone H1° is not a major linker
histone during early mouse development. Instead, somatic
H1 was present throughout this period. Though present in
mature oocytes, somatic H1 was not found on maternal
metaphase II chromatin. Upon formation of pronuclear
envelopes, somatic H1 was rapidly incorporated onto
maternal and paternal chromatin, and the amount of
somatic H1 steadily increased on embryonic chromatin
through to the 8-cell stage. Microinjection of somatic
H1 into oocytes, and nuclear transfer experiments,
demonstrated that factors in the oocyte cytoplasm and the
nuclear envelope, played central roles in regulating the
loading of H1 onto chromatin. Exchange of H1 from
transferred nuclei onto maternal chromatin required
breakdown of the nuclear envelope and the extent of
exchange was inversely correlated with the developmental
advancement of the donor nucleus.
INTRODUCTION
up and down regulation of the expression of specific genes
(Shen et al., 1995; Shen and Gorovsky, 1996; Dou et al., 1999).
A potentially regulatory facet in a number of organisms is the
use of a repertoire of linker histone variants which differ both
in their globular domains, and in modification of the length
and net charge of the C-terminal domain. In the mouse, for
example, there are five somatic variants H1a, H1b, H1c, H1d
and H1e, a testis specific variant H1t, and the variant H1°
which is expressed only in some lineages of differentiated cells
(Franke et al., 1998). In the transition from oocyte to somatic
5S rRNA gene expression during Xenopus embryogenesis
(Wolffe, 1989; Bouvet et al., 1994), somatic histone H1 binds
equally to both oocyte and somatic 5S nucleosomal templates
(Howe et al., 1998) but it selectively represses the oocyte
template through binding to the 3′ end of the nucleosomal
core, resulting in stable positioning of a nucleosome over key
regulatory elements (Sera and Wolffe, 1998). On the somatic
template, H1 binds to the 5′ end of the nucleosomal core,
leaving key promoter elements accessible. In the chicken,
where the six H1 genes encode different H1 protein sequences
Linker histones interact with spacer DNA between adjacent
nucleosomal histone octamer cores. The traditional view that
they are a stoichiometric structural component of chromatin,
with an essentially repressive role in regulating transcription,
has been undergoing revision. In contrast to the structural
and sequence conservation of the core histones, there is
considerable divergence in both sequence and structure among
linker histones. Metazoan linker histones contain a central
globular domain with N- and C-terminal tails, but the
protozoan, Tetrahymena, has a linker histone which contains
only the C-terminal tail (reviewed by Wolffe et al., 1997). The
C-terminal region is rich in basic amino acids, and it is likely
that this tail domain interacts with negatively charged linker
DNA to facilitate chromatin condensation (Ramakrishnan,
1997).
The knockout of histone H1 in Tetrahymena revealed two
important points; histone H1 is not essential for nuclear
assembly or cell survival, and it appears to be involved in both
Key words: Histone H1°, Genome activation, Oocyte, Nuclear
transfer
2898 P. G. Adenot and others
(Nakayama et al., 1993), different protein patterns were
obtained from a series of mutants cell lines, each lacking one
of the H1 genes, indicating that H1 variants may play distinct
roles in the transcriptional regulation of specific genes (Takami
et al., 2000).
A particular feature of early embryogenesis in some animals,
is the absence of somatic linker histones during the initial
cleavage stages. Prior to the mid-blastula transition (MBT) in
Xenopus embryos, an embryonic variant H1M (or B4) replaces
somatic H1 (Smith et al., 1988; Dimitrov et al., 1993). The high
mobility group protein HMG-1, together with the B4 linker
histone, are major components of chromatin within the nuclei
assembled during the incubation of Xenopus sperm chromatin
in Xenopus egg extract (Nightingale et al., 1996). Both proteins
bind to linker DNA but less tightly than somatic H1 (Ura et al
1996), and thus may facilitate rapid cycles of DNA replication.
In Drosophila embryos, an HMG-1 homologue, HMG-D,
replaces somatic H1 until the MBT (Ner and Travers, 1994).
In the sea urchin, maternal cleavage stage histones are present
until the third cell cycle, and the cleavage stage linker histone
has a high homology to Xenopus histone B4 (Mandl et al.,
1997). However, it remains questionable whether this feature
can be extended to early embryogenesis in general, or if it may
instead reflect a situation in which rapid DNA replication
occurs in the absence of transcription from the zygotic genome.
In the mouse, the initial observation by Clarke et al. (1992),
that somatic histone H1 is first detected cytochemically in a
portion of embryos at the 4-cell stage and by the 8-cell stage
in all nuclei in all embryos, suggested that the mouse embryo
followed the early cleavage pattern of the sea urchin, Xenopus
and Drosophila, in maintaining the absence of a somatic linker
histone. The mouse embryo, however, would deviate in two
important ways; somatic H1 appears one full cell cycle after
major activation of the zygotic genome (reviewed by Latham,
1999), and Clarke et al. (1997) subsequently proposed that the
differentiation variant H1°, rather than an embryonic variant,
was the substitute linker histone. In the ovary, the fully grown
oocyte is a highly specialized cell which results from a
differentiation process during oogenesis. From this point of
view, the presence of the differentiation variant H1° in
chromatin could be considered consistent. The amount of H1°
increases during terminal cell differentiation (Rousseau et al.,
1992), and its overproduction results in a decrease of
transcriptional activity (Brown et al., 1997). However, the
immediate future of the differentiated oocyte is to become a
totipotent cell following fertilization or parthenogenetic
activation, and this is difficult to reconcile with the persistence
of H1° in embryonic nuclei beyond activation of the zygotic
genome (Clarke et al., 1997). In studying histones in mouse
oocytes and embryos by radiolabelling coupled to SDS-PAGE,
Wiekowski et al. (1997) showed that nascent linker histone in
the fully grown oocyte and in the embryo up to the 2-cell stage
are synthesized from maternal mRNAs and that they migrated
as somatic H1 during SDS-gel electrophoresis, suggesting that
somatic H1 may be present during this developmental period.
In this study, we have examined whether mouse embryos
deviate from the early cleavage pattern of the sea urchin,
Xenopus, and Drosophila, by maintaining somatic linker
histone in chromatin. Using antibodies that recognize somatic
H1 (Dimitrov and Wolffe, 1996), mouse phosphorylated H1
(Chadee et al., 1995), and mouse H1° (Gorka et al., 1998), we
show that somatic histone H1 was present in chromatin until
nuclear breakdown during meïotic oocyte maturation, and then
reassembled onto chromatin following pronuclear formation at
the onset of embryogenesis. Histone H1° was not detected on
chromatin during this developmental period. The absence of
H1 on maternal metaphase II chromosomes, contrasted with its
presence on chromosomes at the first mitosis and a weak
presence in sperm chromatin. Microinjection of somatic H1
into oocytes did not result in staining of maternal
chromosomes, but in nuclear transfer experiments, we
observed that somatic H1 could be loaded onto maternal
chromosomes only when the transferred nucleus lost its nuclear
membrane and formed prematurely condensed chromosomes
(PCC). The extent of transfer of H1 to maternal chromosomes,
and its removal from PCC, appeared to be mediated by
factors in the oocyte cytoplasm, but also depended on the
developmental stage of the transferred nucleus.
MATERIALS AND METHODS
Collection of embryos and oocytes
Female C57/CBA mice, 6-8 weeks old, were superovulated with
intraperitoneal injections of 5 i.u. of pregnant mare serum (PMS;
Folligon, Intervet), followed 46-48 hours later with 5 i.u. human
chorionic gonadotropin (hCG; Chlorulon, Intervet). To recover mature
oocytes in metaphase II (MII), superovulated females were sacrificed
15 hours post-hCG (phCG). To obtain fertilized oocytes, females were
caged with C57/CBA males immediately after hCG injection, and
embryos were collected at the one-cell stage. Oocytes and embryos
were incubated after collection in 0.5% hyaluronidase (Sigma) in PB1
for 1-2 minutes at 37°C to remove cumulus cells, washed extensively
in PB1, and then returned to culture in M16 medium under 5% CO2
in air, until fixation. To recover immature oocytes at prophase I,
female C57/CBA mice, 11-13 weeks old, were superovulated with
intraperitoneal injections of PMS as described above. Ovaries were
removed 45 to 50 hours after injection, and transferred to PB1
prewarmed at 37°C. Fully grown oocytes were freed from peripheral
cells by gentle pipetting, and either fixed immediately, or returned to
culture to be fixed during nuclear maturation.
Nuclear transfer procedures
Hybrid cells were reconstructed by electrofusion between MII oocytes
and either embryonic, or somatic cells. Following fusion, transferred
nuclei condense prematurely into chromosomes if the oocyte is not
already parthenogenetically activated at the time of cell fusion
(reviewed by Campbell, 1999). MII oocytes used as recipient cells
were 16-20 hours phCG at the time of fusion. Blastomeres from mid
(43 hours phCG) and late (50 hours phCG) 2-cell embryos, from late
4-cell embryos (67 hours phCG), and from early 8-cell embryos (72
hours phCG) cleaved during the previous hour, were used as sources
of embryonic nuclei. Cumulus cells freshly removed from MII
oocytes, as described above, were the source of somatic nuclei.
Two nuclear transfer procedures were performed according to the
size of the donor cell. Donor cumulus cells were washed extensively
in PB1 and then introduced beneath the zona pellucidae of MII
oocytes. Resulting pairlets were washed in 0.3 M mannitol, placed in
the same solution into a fusion chamber between platinium electrodes,
and then subjected to two DC pulses of 1.5 kV/cm, 100 microseconds
each. When the donor cell was a blastomere, the zona pellucida was
removed from MII oocytes and embryos by gentle pipetting following
a treatment with 0.25% Pronase (Sigma) in PB1 for 1 minute at 37°C.
Embryos were then incubated 15 minutes at 37°C in PBS without
Ca2+ and Mg2+ (Gibco) to dissociate blastomeres. MII oocytes and
blastomeres were agglutinated in 150 µg/ml phytohaemagglutinin
Linker histone H1 in early mouse embryogenesis 2899
(Sigma) in PB1, for 3 minutes at 37°C. Aggregated pairs were washed
in 0.28 M mannitol and electrofused in this solution. Electrofusion
was done as above except that each pulse lasted 60 microseconds.
Following electrofusion, both oocyte-blastomere and oocyte-cumulus
cell pairlets were rinsed in PB1 and cultured in M16 under 5% CO2
in air. Fusion between the oocyte and the blastomere occured 5 to 40
minutes post pulse, and resulting hybrid cells were fixed 1.5 to 2 hours
post fusion. The time of fusion between the oocyte and the cumulus
cell could not be precisely determined because of the small size of a
cumulus cell. Therefore, oocyte-cumulus cell pairlets were fixed 1.5
to 2 hours after the electrical pulses. To control for effects of the
experimental procedure, hybrid cells were also reconstructed from
two MII oocytes.
Microinjection of oocytes
Microinjection of somatic histone H1 was done essentially as
described (Lin and Clarke, 1996). Calf thymus histone H1
(Boehringer) was dissolved in water at 1 mg/ml. Microinjection into
MII oocytes was carried out using a Nikon inverted microscope
equiped with Narishige micromanipulators and an Eppendorf
microinjector. About 1-5 pl of histone H1 solution was injected per
oocyte. Following injection, MII oocytes were returned to culture for
2 hours before fixation. The same injection into the cytoplasm of 1cell embryos induced an accumulation of linker histones in pronuclei
within 15 minutes (Lin and Clarke, 1996).
Antibodies
Mouse histone H1° was detected with a monoclonal antibody raised
against ox liver H1° (clone 34B10H4, a generous gift from Dr S.
Kochbin) and characterized by Dousson et al. (1989). Mouse somatic
histone H1 was detected with two antisera; one raised against Xenopus
somatic H1 (Dimitrov and Wolffe, 1996), and the other against
phosphorylated isoforms of Tetrahymena H1 (Lu et al., 1994).
Secondary antibodies were FITC-conjugated goat anti-rabbit and antimouse IgG (Sigma, 1:400 final dilution).
Confocal immunofluorescence microscopy
Oocytes, embryos, hybrid cells and differentiated cells were fixed in
2.5% paraformaldehyde for 20 minutes at room temperature. They
were incubated for 30 minutes at 37°C in a blocking solution
containing 10% foetal calf serum (FCS) and 0.2% Triton X-100 in
PBS. Subsequent manipulations were performed in PBS/2% FCS/0.1%
Triton X-100 solution. Nuclear antigens were detected by indirect
immunofluorescence: cells were incubated with the primary antibody
overnight at 4°C, and washed extensively before incubation for 1 hour
at 37°C with the second antibody. Chromatin was stained for 30
minutes with 10 µg/ml propidium iodide (Sigma). Cells were mounted
on well-slides in Vectashield (Vector Laboratories) containing the same
concentration of propidium iodide, and were observed under a confocal
laser scanning microscope (Carl Zeiss, CLSM 310).
Cell lysates, chromatin preparation and western blot
analysis
Cell lysates were prepared in denaturing buffer L (50 mM Tris-HCl,
pH 6.8, 10% glycerol, 2% sodium dodecyl sulfate (SDS), 1% 2mercaptoethanol). Chromatin was prepared as described by
Wiekowski and DePamphilis (1993). Proteins were electrophoresed
in denaturing 15% polyacrylamide-SDS gels and blotted onto
nitrocellulose. Immunodetection by primary antibodies was revealed
by a peroxidase-labelled immunoglobulin antiserum and enhanced
chemiluminescence (Super Signal ULTRA, Pierce).
RESULTS
The reactivity of anti-histone antibodies was first examined on
mouse linker histones in cell lysates and in fixed cells. The
antibody raised against Xenopus somatic H1 specifically
recognized mouse somatic H1 on immunoblot (Fig. 1A, antiH1 panel), and by immunostaining (Fig. 1B, anti-H1 panel), it
gave an intense nuclear staining in mouse erythroleukemia
(MEL) cells and in mouse blastula cells which is consistent
with the H1 pattern reported in previous immunocytochemical
studies (Clarke et al., 1992; Stein and Schultz, 2000). The
antibody raised against phosphorylated H1 (H1P) in
Tetrahymena, which has been demonstrated to recognize only
one of the mouse H1P subtypes (histone H1b; Chadee et al.,
1995), reacted with one immunoreactive protein, present only
in the mouse cell lysate (Fig. 1A, anti-H1P panel), and
migrating at the position of mouse H1P (Chadee et al., 1995).
By immunostaining (Fig. 1B, anti-H1P panel), this antibody
recognized a nuclear protein in MEL cells and in mouse
blastula cells, with a variable staining level in the nucleus
during interphase, and a much more intense staining on
chromatin during mitosis. At this latter stage, a higher
cytoplasmic staining was also observed. This H1P staining
pattern is consitent with what is known about H1P levels in
mammalian proliferative cells (Roth and Allis, 1992; Chadee
et al., 1995; Bleher and Martin, 1999). The antibody raised
against ox liver H1°, which has been demonstrated to
specifically recognize an epitope in the region of amino acids
20 to 30 in murine H1° on immunoblots (Gorka et al., 1998),
reacted strongly with a protein in mouse cell lysates (Fig. 1A,
anti-H1° panel), migrating at the position of H1° (Djondjurov
et al., 1983), and slightly with somatic H1. By
immunostaining, we observed a clear staining in nuclei of MEL
cells (Fig. 1B, anti-H1° panel, left column), where histone H1°
is present in a substantial amount (Helliger et al., 1992; Gorka
et al., 1998), and an absence of staining in nuclei of mouse
STO fibroblasts (Fig. 1B, anti-H1° panel, right column),
indicating that the anti-ox liver H1° antibody specifically
recognized H1° but not H1 in fixed cells.
Somatic histone H1 is present in the nucleus of
mouse oocytes and early cleavage embryos
The presence of somatic H1 in oocytes and embryos of early
cleavage stages was first studied by immunoblot, using the
antibody raised against Xenopus somatic H1. Following
autoradiographic exposure of several hours, immunoreactive
proteins, migrating as somatic histone H1 in the blastocyst
embryo (Fig. 2, lane 5), were detected in lysates prepared from
1100 MII oocytes or 1-cell embryos (Fig. 2, lanes 1 and 2,
respectively). A very faint signal was obtained with the same
number of germinal vesicle (GV) stage oocytes (not shown).
At the 2-cell and 4-cell stages when somatic H1 has been
reported to be present (Clarke et al., 1992; Wiekowski et al.,
1997; Stein and Schultz, 2000), this protein was detected in
lysates prepared from only 300 to 400 embryos (Fig. 2, lanes
3 and 4, respectively). Thus, somatic H1 was present in mouse
oocytes and 1-cell embryos, and it increased substantially by
the 2-cell stage.
Histone H1 immunostaining of chromatin was observed in
GV oocytes (Fig. 3A,E), and in embryos following pronuclear
formation at the 1-cell stage (Fig. 3J-L, and N-R). No H1
staining was found on maternal chromatin between GV
breakdown (Fig. 3B,F and C,G) and pronuclear formation in
fertilized or parthenogenetically activated oocytes (not shown).
H1 staining of chromatin was of low intensity in the 1-cell
2900 P. G. Adenot and others
Fig. 1. Recognition of mouse histones by anti-histone antibodies.
(A) Immunoblotting of lysates prepared from mouse erythroleukemia
(MEL) cells or mouse HC11 cells. Equal amounts of lysates were
loaded onto the same gel, together with calf thymus histone H1,
subjected to SDS-PAGE, and blotted onto nitrocellulose. The blot was
cut and each part was incubated separately with the anti-Xenopus
somatic H1 antibody (anti-H1 panel), the anti-Tetrahymena
phosphorylated H1 antibody (anti-H1P panel), or the anti-ox liver H1°
antibody (anti-H1° panel). Arrows indicate the migrating position of
the H1 doublet. (B) Confocal images of immunostained and DNA
stained mouse blastula cells (anti-H1 and anti-H1P panels, right
columns), mouse STO fibroblasts (anti-H1° panel, right columns), and
proliferative MEL cells (left columns) immunolabelled with the antiXenopus somatic H1 antibody (anti-H1 panel), the anti-Tetrahymena
phosphorylated H1 antibody (anti-H1P panel), or the anti-ox liver H1°
antibody (anti-H1° panel). Bar, 30 µm; relative magnification = 1
(MEL cells, anti-H1 and anti-H1P panels), 1.4 (MEL cells, anti-H1°
panel), 1.1 (blastula cells), 0.9 (STO cells).
embryo, though several intense spots located at the periphery
of prenucleolar bodies were transiently observed during the
few hours which followed pronuclear formation (Fig. 3J,N). In
Fig. 2. Immunodetection of somatic H1 in the mouse oocyte and
embryo with the anti-Xenopus H1 antibody. Lysates were prepared
from 1125 MII oocytes (1), 1153 one-cell embryos at 24-26 hours
phCG (2), 397 two-cell embryos at 40-45 hours phCG (3), 297 fourcell embryos at 56-66 hours phCG (4), and 209 blastocysts (5).
Autoradiographic exposure was for 21 hours (1-2), 23 hours (3-4) or
10 seconds (5). Calf thymus histone H1 served as a marker for
position of the H1 doublet (arrows).
contrast to meïotic oocyte chromosomes (Fig. 3C,G), somatic
histone H1 was present at very low levels in decondensing
sperm chromatin at fertilization (Fig. 3C,G), and on zygotic
chromosomes at the first mitosis (Fig. 3K,O). When embryos
were fixed at the mid 2-cell stage, nuclear H1 staining intensity
Linker histone H1 in early mouse embryogenesis 2901
Fig. 3. Confocal images of mouse oocytes and early
embryos immunolabelled with the anti-Xenopus somatic H1
antibody (A-C, J-L, Q), or with the pre-immune serum
(D,I), and DNA stained (second, fourth and bottom rows).
Detector sensitivity was the same for A, B and L, was 3-fold
more sensitive for J, K and the insert in B, was 6-fold more
sensitive for C, I, and 8-fold more sensitive for D. Images
A-P were obtained with a ×63 oil immersion objective and
images Q, R, with a ×16 oil immersion objective.
(A,E) Immature oocyte; arrow designates the germinal
vesicle. (B,F) Oocyte after GV breakdown; insert, the same
oocyte at higher detector sensitivity. (C,G and
D,H) Fertilized eggs; arrow indicates the position of the
very faintly staining sperm head. (I,M and J,N) One-cell
pronuclear embryo at 20-22 hours phCG. Dense spots of
histone H1 were present at the periphery of prenucleolar
bodies (arrowheads), and non-specific staining was found in
the sperm tail (arrow). (K,O) One-cell embryos at mitosis.
(L,P) Two-cell embryo at 43 hours phCG; a peripheral
enrichment of histone H1 in nuclei was observed and was not related to the presence of condensed chromatin in this region. (Q,R) One-cell (a),
two-cell (b), four-cell (c) and eight-cell (d) embryos observed in the same field. The quantity of histone H1 in nuclei increased from the one-cell
stage to the eight cell stage. Bar, 30 µm. Relative magnification = 1 (A-P), 0.3 (Q,R).
had increased strongly, and an enriched perinuclear staining
was sometimes observed (Fig. 3L,P). At subsequent cleavage
stages, nuclear H1 staining intensity increased continuously
(Fig. 3Q,R). Therefore there was a clear correlation between
the increasing immunostaining of nuclei of individual embryos
from the 1- to 8-cell stage, and the decreased number of
embryos required to obtain a signal by immunoblotting as
cleavage stage development progressed.
As an additional test to confirm the presence of somatic
histone H1 in the chromatin of GV oocytes and early embryos,
2902 P. G. Adenot and others
Fig. 4. Confocal images of
immunostained (top row) and
DNA stained (bottom row)
mouse oocytes and early
embryos immunolabelled with
the anti-Tetrahymena
phosphorylated H1 antibody
(B-E) or with the pre-immune
serum (A). (A,F and
B,G) Mouse oocytes at the
germinal vesicle stage
surrounded by cumulus cells;
arrow designates the germinal vesicle and arrowhead a cumulus cell in mitosis. (C,H) 1-cell embryo 23 hours phCG. (D,I) 2-cell embryo 46
hours phCG. (E,J) 4-cell embryo 62 hours phCG. Bar, 30 µm.
immunostaining was done with the antibody raised against
phosphorylated H1 in Tetrahymena. Nuclei were faintly stained
in oocytes (Fig. 4B,G) and more clearly labelled in 1-cell (Fig.
4C,H), in 2-cell (Fig. 4D,I) and in 4-cell (Fig. 4E,J) embryos.
No chromatin staining was detected in oocytes from GV
breakdown to the meïotic arrest at metaphase II (not shown).
These results confirm the presence of histone H1 in chromatin
of oocytes and early embryos. Thus, we conclude that somatic
histone H1 is located in embryonic nuclei as early as the 1-cell
stage, and that substantial nuclear import has taken place by
the 2-cell stage, when major zygotic genome activation occurs.
the experimental procedure, as it was never observed following
fusion between two MII oocytes (Fig. 5I,L). Interestingly,
somatic histone H1 was undetectable on oocyte chromatin when
the transferred embryonic nucleus did not form PCC (not
shown), nor following microinjection of calf thymus H1 into the
MII oocyte (not shown). Taken together, these results show that
MII oocytes contain cytoplasmic activities which remove histone
H1 from chromatin when a nuclear envelope is absent. We
conclude that histone H1 is undetectable on oocyte MII
chromosomes because it is removed from chromatin during
oocyte nuclear maturation.
Histone H1 release from chromatin following nuclear
transfer in MII oocytes
The lack of detection of somatic histone H1 on meïotic oocyte
chromosomes, and its presence on zygotic chromosomes,
suggest that somatic histone H1 is removed from chromatin
during nuclear maturation in oocytes. To test this idea, we
performed nuclear transfer experiments to simultaneously
observe both MII chromosomes and embryonic or somatic
prematurely condensed chromosomes (PCC) in the same oocyte
cytoplasmic environment. Resulting hybrid cells were
immunolabelled with the anti-Xenopus H1 antibody (Fig. 5).
Following the transfer of a cumulus cell nucleus, the PCC and
oocyte chromosomes showed no staining for histone H1 (Fig.
5A,D). In contrast, both sets of chromosomes were positively
labelled following the transfer of embryonic nuclei. In these
latter hybrid cells, the PCC were less intensely stained than
corresponding zygotic chromosomes at the same cleavage stage
(not shown), indicating that histone H1 was also released from
chromatin. The PCC formed from mid (Fig. 5B,E and 5C,F) or
late (not shown) 2-cell embryonic nuclei remained generally
distinguishable by anti-H1 staining, while PCC originating from
late 4-cell (Fig. 5G,J), or early 8-cell (Fig. 5H,K) embryonic
nuclei did not. Thus, when using later stage embryonic nuclei,
relatively little histone H1 remained on chromatin, and
numerous foci of histone H1 were observed near the PCC. We
also observed that oocyte chromosomes exhibited more intense
H1 staining when the PCC also conserved high H1 content, and
that the uptake of histone H1 by oocyte chromosomes was not
at all correlated with the amount of H1 lost from the PCC;
though 4-cell and 8-cell embryonic nuclei had higher H1 content
than 2-cell nuclei, hybrids formed from the former group
exhibited both PCC and oocyte chromosomes with very low H1
staining. H1 staining of oocyte chromosomes did not result from
Histone H1° is not detected as a replacement linker
histone in mouse oocytes and embryos
The low H1 staining of chromatin in the GV oocyte and the 1cell embryo may reflect the presence of a substitute linker
histone during this developmental period. Since the
differentiation variant H1° has been proposed as a candidate for
this replacement, based on cytochemical observations in murine
oocytes and early embryos with an antibody against the related
chicken H5 protein, combined with RT-PCR analysis (Clarke et
al., 1997), we investigated this proposal using the antibody
raised against ox liver H1°, which specifically recognizes murine
H1° (Gorka et al., 1998). We did not detect any labelling of GV
stage oocytes (Fig. 6A,E), in chromosomes of MII oocytes (not
shown), in pronuclei of 1-cell embryos (Fig. 6B,F), nor in nuclei
of 2-cell (Fig. 6C,G), or 4-cell (Fig. 6D,H) embryos. This was
in distinct contrast to the clear detection of H1° in MEL cells
(Fig. 1B). Thus, we conclude that H1° is not the predominant
linker histone during early mouse development.
DISCUSSION
A striking feature of early embryogenesis in the sea urchin,
Drosophila, and Xenopus is the use of specific cleavage stage
linker histones or high mobility group proteins in place of
somatic histone H1. In the latter two organisms, somatic
histone H1 begins to accumulate near the MBT, a time when
transcription from the zygotic genome begins. In Xenopus
embryos, the presence of somatic H1 has been shown to
regulate the switch from transcription of oocyte 5S rRNA
genes to somatic 5S genes (Sera and Wolffe, 1998) and to
restrict the competence of ectodermal cells to differentiate into
mesoderm (Steinbach et al., 1997). This modification of linker
Linker histone H1 in early mouse embryogenesis 2903
Fig. 5. Distribution of histone
H1 following nuclear transfer
and premature chromosome
condensation. Hybrid cells
were reconstructed by
electrofusion between MII
oocytes and cumulus cells
(A,D), or embryonic
blastomeres at the mid 2-cell
(B,C,E,F), late 4-cell (G,J), or
early 8-cell (H,K) stages. To
control for effects of the
fusion protocol, MII oocytes
were also fused together (I,L).
Top and third rows: H1
immunostaining. Second and
fourth rows: counterstaining of
DNA with propidium iodide.
In histone H1 immunostained
micrographs, arrows designate
chromosomes of the oocyte
(long arrow) and the
transferred nucleus (short
arrow). All images were
obtained at the same detector
sensitivity. Inserts in G and H
show a magnified view where
several foci of histone H1
were observed near
prematurely condensed
chromosomes. Arrow in L
indicates two sets of
chromosomes corresponding
to telophase II in the oocyte
hybrid cell. Bar, 25 µm.
Fig. 6. Confocal images of
immunostained (top row) and
DNA stained (bottom row)
mouse oocytes and early
embryos immunolabelled with
the anti-ox liver H1° antibody.
(A,E) Mouse oocytes at the
germinal vesicle stage.
(B,F) 1-cell embryo 23 hours
phCG. (C,G) 2-cell embryo 46
hours phCG. (D,H) 4-cell
embryo 62 hours phCG. Bar,
30 µm.
histone types during early development is somewhat
reminiscent of nature’s experimentation with the replacement
of sperm nuclear basic proteins (SNBPs) during the process of
spermatogenesis. Ausio (1999) has recently reviewed a
proposal of the derivation of protamines (P) from a histone H1
precursor (H1), via protamine-like intermediates (PL),
2904 P. G. Adenot and others
concluding that the basic evolutionary H1 → PL → P
progression connecting these basic proteins has occured
repeatedly on many occasions during metazoan evolution. The
apparent random distribution of SNBPs throughout the animal
kingdom would be the net result of these events superimposed
on a background of multiple reversions. Whether any
evolutionary pattern in the use of embryonic variants of linker
histones exists, is for the moment, not clear.
Somatic histone H1 is present during the first
cleavage stages of mouse development
During the last decade, it has been thought that the mouse
embryo followed the early cleavage pattern of the sea urchin,
Xenopus, and Drosophila, in maintaining the absence of a
somatic linker histone, because somatic H1 was not detected
on chromatin until the mid 4-cell stage with an anti-rat somatic
H1 antibody (Clarke et al., 1992). Using an antibody raised
against Xenopus somatic H1 (Dimitrov and Wolffe, 1996),
we have demonstrated by western blotting and
immunofluorescence, that somatic H1 was present in the
unfertilized mouse egg, in 1-cell pronuclei, 2-cell nuclei and
in 4-cell nuclei. The amount was low in pronuclei and
increased through to the 8-cell stage.
The fact that we detect somatic H1, where Clarke et al. (1992)
did not, may result from the differential sensitivity of the
antibody reagents and of detection methods. A mouse oocyte
contains approximatively 60 pg of histone (Wassarman and
Mrozak, 1981). Therefore, in our immunoblots, somatic H1 in
the unfertilized egg was detected in a lysate containing about 70
ng of total histone, whereas Clarke et al. were unable to detect
H1 in a lysate containing twice as much oocyte histone. A more
recent immunocytochemical study in the early mouse embryo
(Stein and Schultz, 2000) showed that the anti-rat H1 antibody
used in the Clarke et al. study readily detects the presence of
somatic H1 on chromatin by the late 2-cell embryonic stage, one
cleavage stage earlier than previously reported (Clarke et al.,
1992). The differential detection of somatic H1 may also be
related to linker histone composition in the early mouse embryo.
Somatic H1 subtypes migrate as two prominent bands during
SDS-gel electrophoresis. Wiekowski et al. (1997) found that the
two migrating H1 variants are synthesized in the fully grown
oocyte, with the faster migrating H1 variant being predominant.
They also demonstrated that histone H1 synthesis, which is
arrested when the oocyte undergoes nuclear breakdown and
nuclear maturation (Wassarman and Letourneau, 1976), resumes
in the embryo from the late 1-cell/early 2-cell stage, but only the
faster migrating H1 variant was detectable in the 2-cell embryo.
In comparing the two antibodies by western blotting of mouse
spleen and HC11 cells preparations, we found that the faster
migrating H1 variant was predominant with the anti-Xenopus
antibody, whereas the slower migrating H1 variant was
predominant with the anti-rat antibody (data not shown). This
may explain the increased sensitivity of the anti-Xenopus H1
antibody in detecting somatic H1 during early murine
embryogenesis.
The differentiation variant H1° is not a predominant
linker during the first cleavage stages of mouse
development
The low content of somatic H1 in pronuclei means that we can
not formally exclude the presence of a substitute linker histone
in earliest cleavage stage mouse embryos. Previous
immunocytochemical observations, with an antibody that
recognizes chicken histone H5 and ox liver histone H1°, but
not chicken H1 on immunoblot (Allan et al., 1982), suggested
that the predominant linker histone in post-natal murine
oocytes and early embryos was immunologically related to the
differentiation variant H1° (Clarke et al., 1997). Using an
antibody raised against ox liver H1° (Dousson et al., 1989),
which recognizes murine H1° on immunoblot (Gorka et al.,
1998), and an anti-Xenopus somatic H1 antibody which
recognizes mouse somatic H1, we detected both H1° and
somatic H1 by immunofluorescence in MEL nuclei, and only
somatic H1 in nuclei of mouse oocytes and early embryos. As
MEL nuclei contain a substantial amount of H1° (Helliger et
al., 1992; Gorka et al., 1998), we conclude that the
differentiation variant H1° is not a predominant linker histone
on chromatin during this developmental period. We have also
been unable to detect any proteins recognized by an antibody
directed against the Xenopus embryonic variant B4
(unpublished observations). The high abundance of xHMG1
and HMG-D in Xenopus and Drosophila cleavage stage
embryos, followed by a sharp reduction at the MBT, suggests
that they may in part substitute somatic H1 functions (Dimitrov
et al., 1993, 1994; Ner and Travers, 1994), but this is not the
case in mouse embryos, as the profile of HMG1 abundance
(Spada et al., 1998), follows precisely that described for
somatic H1 in this study.
What regulates linker histone association with
chromatin during early mouse embryogenesis?
Metabolic radiolabelling studies have shown that histone H1 is
synthesized in the fully grown mouse oocyte (Wassarman and
Letourneau, 1976; Wiekowski et al., 1997). In vivo, we
observed that somatic histone H1 remained in the germinal
vesicle of fully grown oocytes, in contrast to previous reports
(Clarke et al, 1997), and was absent on maternal chromatin
from GV breakdown through to pronuclear formation
following fertilization or parthenogenetic activation. Which
activities might be involved in the removal of histone H1 from
oocyte chromatin? The interaction of histone H1 with
chromatin is regulated through phosphorylation. It has been
noted that phosphorylation of H1 is increased in highly
proliferative cells, reduced in quiescent cells, and that
phosphorylation levels are maximal, just prior to, or at
metaphase, with a rapid decrease thereafter (reviewed by Roth
and Allis, 1992). These observations led to the notion that H1
phosphorylation is important in mitotic chromosome
condensation. Meïotic reinitiation and nuclear maturation in
the mouse oocyte is a complex process which requires the
gradual activation of the p34cdc2 H1 kinase (Gavin et al.,
1994). This activity first increases 2-fold at GV breakdown,
and then 8-fold in a protein synthesis-dependent manner as the
oocyte progresses to metaphase I. The present study reveals
that the removal of histone H1 from oocyte chromatin is
coincident with minor p34cdc2 H1 kinase activation. Although
phosphorylation should weaken interactions between
nucleosomal DNA and linker histone (Hill et al., 1990; Talasz
et al, 1998), the removal of histone H1 from oocyte chromatin
can not be attributed solely to linker histone phosphorylation
because H1 remains absent from chromatin following p34cdc2
H1 kinase inactivation in fertilized or parthenogenetically
Linker histone H1 in early mouse embryogenesis 2905
activated oocytes. It is now known that H1 phosphorylation is
not necessary for mitotic chromosome condensation (Guo et
al., 1995; Ajiro et al., 1996), and that histone H1 itself is not
required for nuclear assembly (Dasso et al., 1994) or for
chromosome condensation in Xenopus (Ohsumi et al., 1993)
or Tetrahymena (Shen et al., 1995). Since a similar amount of
cells was necessary to detect somatic H1 by western blotting
in the MII oocyte and the 1-cell embryo, this suggests that
linker histone H1 is not required to maintain chromatin
condensation during oocyte nuclear maturation.
Substantial chromatin remodelling occurs in the mouse
zygote (Perreault, 1992; Nonchev and Tsanev, 1990; Adenot et
al., 1997). A small, localized distribution of somatic H1
remains in mature sperm chromatin (Pittoggi et al., 1999), and
a weak somatic H1 staining of sperm chromatin was observed
immediately following fertilization. More substantial
accumulation of somatic H1 on maternal and paternal
chromatin began after formation of pronuclear envelopes,
though this clearly preceded the detection of nascent linker
histones (Wiekowski et al., 1997), suggesting that the nuclear
envelope may play a role in regulating the loading of H1 onto
chromatin in the mouse zygote. It is known that in contrast to
core histones, a relatively large pool of H1 is found in the
cytoplasm of both proliferating and quiescent cells (Zlatanova
et al., 1990). Using a digitonin permeabilization assay system,
Kurz et al. (1997) have shown that nuclear transport of H1
histones meets the criteria of a nuclear localization signal
mediated-process, and it has been found recently that import
of H1 into the nucleus requires the cytoplasmic assembly of a
complex including H1, importin β, and importin 7, and the
presence of functional nuclear pore complexes (Jäkel et al.,
1999).
It has been demonstrated that microinjection of somatic H1
into 1-cell embryos results in its rapid uptake onto pronuclear
chromatin (Lin and Clarke, 1996; Stein and Schultz, 2000). In
this study, microinjection of somatic H1 into MII oocytes
resulted in no uptake of H1 onto MII chromosomes. When
performing nuclear transfer we noted that histone H1 remained
on chromatin in the transferred nucleus and was never found on
MII chromosomes, provided that the nuclear envelope remained
intact (data not shown). If nuclear envelope breakdown
occurred, leading to the formation of PCC, the presence of H1
on both PCC and MII chromosomes depended on the
developmental stage of the donor nucleus. When somatic
cumulus cell nuclei were transferred, H1 was removed from
PCC and was not detected on MII chromosomes. When 8-cell
or 4-cell embryonic nuclei were transferred, foci of H1
remained in the vicinity of PCC, and very weak H1 staining was
found on MII chromosomes. However, when a 2-cell nucleus
was transferred, some H1 remained on the 2-cell derived PCC
and was also observed on MII chromosomes. These results
show that the oocyte contains activities which remove somatic
H1 from chromatin. There are two potential explanations for the
dependance of the extent of this removal on the developmental
stage of the donor nucleus. It is possible that the limited release
of histone H1 from embryonic nuclei simply results from the
higher dilution of oocyte cytoplasmic activities upon cell fusion,
since a blastomere is much larger than a cumulus cell. The other
possibility is that nuclear factors present in the 2-cell embryo,
and to a much lesser extent in 4- and 8-cell embryos, are able
to at least partially reverse the removal of somatic H1 by the
oocyte. A clear weakness of the simple dilution argument,
however, is that in this case, the microinjection of H1 into
oocytes should also saturate H1 removal agents in the oocyte
and result in H1 loading onto MII chromosomes.
In a study of remodelling of somatic nuclei in Xenopus egg
extracts, Dimitrov and Wolffe (1996) have shown that histone
H1° is quantitatively removed from chromatin, as is somatic
H1 to a lesser extent, and that this process is mediated by egg
nucleoplasmin. In the Xenopus oocyte, nucleoplasmin
accumulates in the germinal vesicle until nuclear breakdown
(Litvin and King, 1988). At fertilization, it removes sperm
specific basic proteins to deposit histones H2A/H2B onto
sperm chromatin (reviewed by Laskey et al., 1993). This
molecular chaperone remains present in nuclei during early
embryogenesis, and then becomes undetectable in adult tissues
(Bürglin et al., 1987; Litvin and King, 1988). It is likely that a
molecular chaperone similar to Xenopus egg nucleoplasmin
exists in the mouse oocyte and during the first cleavage stages
of early embryogenesis, based on what occurs to mouse sperm
chromatin following its incubation in Xenopus oocyte extracts
(Montag et al., 1992), or its microinjection into maturing
oocytes (McLay and Clarke, 1997), immature oocytes, or early
embryos (Maeda et al., 1998). We have recently found a protein
with molecular and antigenic properties similar to Xenopus egg
nucleoplasmin that is present in the mouse oocyte and has an
expression pattern restricted to the early cleavage stages of
mouse development (unpublished observations). Thus, it is
tempting to speculate that removal of H1 from chromatin
during oocyte nuclear maturation is also mediated by a
nucleoplasmin-like protein.
Conclusions
In contrast to the absence of somatic H1 during the first
cleavage stages of sea urchin, Xenopus, and Drosophila
development, we find that somatic H1 is present on chromatin
in mouse embryos as soon as pronuclei have formed at the 1cell stage. We have also observed somatic H1 in 1-cell
pronuclei of cow embryos (unpublished observations)
suggesting that this may be a common feature in mammalian
embryos. The evidence in this study also directly contradicts
the hypothesis (Clarke et al., 1997) that the differentiation
variant H1° serves as a replacement linker histone during early
mouse embryogenesis. Wiekowski et al. (1997), have shown
that new synthesis of histone H1 begins in the late 1-cell
embryo, indicating that the H1 we observe in early pronuclei
and on immunoblots of MII oocytes is of maternal origin.
Through nuclear transfer experiments, and microinjection of
somatic H1, we provide evidence that the absence of histone
H1 on MII chromosomes, despite its presence in the oocyte, is
due to activities in the oocyte cytoplasm which remove somatic
H1 from chromatin. Both the normal developmental profile of
H1 on chromatin, and the results of the nuclear transfer
experiments, demonstrate the central role of the nuclear
envelope in regulating the loading of H1 onto chromatin during
early development.
In Xenopus and mouse embryos, a basal transcription
machinery exists prior to activation of the zygotic genome
(Newport and Kirschner, 1982; Bellier et al., 1997). However,
in Xenopus eggs, a limited translation of stored maternal
mRNAs coding for key components of the transcription
machinery (Veenstra et al., 1999), and a deficiency in the
2906 P. G. Adenot and others
activity of transcriptional activators (Almouzni and Wolffe,
1995), together with the required titration of suppressor
components by DNA (Newport and Kirschner, 1982), may
limit transcription during the early cleavage period. In contrast,
the mouse embryo contains transcription factors as early as the
1-cell stage, though they are in a limited amount (reviewed by
Latham, 1999), and endogenous transcription can readily begin
in pronuclei (Aoki et al., 1997). Plasmid microinjection
experiments into 1-cell pronuclei have revealed that very first
cleavage stage murine embryos are permissive for expression
from a variety of promoters (Bonnerot et al., 1991).
DePamphilis and colleagues (reviewed by Latham, 1999) have
shown that enhancers are dispensable for expression of
episomal templates in 1-cell embryos, but that enhancers are
required to avoid repression of weak promoters in 2-cell
embryos. Global endogenous transcription rates increase
sharply in mouse embryos between the 1-cell and the 4-cell
stages. During this same period we show that the nuclear
content of somatic histone H1 is also increasing dramatically.
If somatic histone H1 is considered as a general structural
repressor of transcription, why is it increasing in content at the
same time as global transcription increases? In contrast to
mammals, Drosophila and Xenopus do not appear to
extensively use methylation as a means of essentially
repressing gene expression. Murine gametes are heavily
methylated and there is genome wide demethylation from the
8-cell stage until minimum methylation is acheived in
blastocysts (Monk et al., 1987). Putting all of the data together,
it may be that in the early mouse embryo, methylation serves
as a general repressor, while histone H1 accumulates
progressively during early cleavage murine embryogenesis. If
somatic H1 is considered as a more selective regulator as in
Xenopus (Wolffe et al., 1997), the progressive accumulation of
somatic H1 during early cleavage stages may participate in the
more controlled transcription that takes place from the 2-cell
stage onward in mouse embryos.
We thank Patrick Chesné, Yvan Mercier, and Bertrand Nicolas,
INRA, for help in nuclear transfer experiments, histone
microinjection, and photomicrograph production, respectively.
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