1 Departments of Obstetrics and Gynecology and Biology, McGill University,
Montreal, Quebec, Canada H3A 1A1
2 Department of Medicine, McGill University, Montreal, Quebec, Canada H3A
1A1
3 Department of Biochemistry and Biophysics and Program in Molecular Biology and
Biotechnology, University of North Carolina, Chapel Hill, NC 27599, USA
Present address: Stanford University School of Medicine, Department of
Genetics, Stanford, CA 94305-5163, USA
Author for correspondence (e-mail:
hugh.clarke{at}muhc.mcgill.ca)
Accepted 24 August 2002
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Summary |
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Key words: SLBP, Histone mRNA, Mouse, Oocyte, Embryo, Translational control, Cell cycle
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Introduction |
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The expression of SLBP is tightly regulated during the somatic cell cycle
(Whitfield et al., 2000). It
is not detectable in G1, but begins to accumulate at the G1/S transition. It
remains abundant during S-phase, and then declines rapidly during G2. SLBP
remains undetectable until the subsequent G1/S transition. Regulation does not
appear to occur at the level of transcription, as the amount of SLBP mRNA
varies little during the cell cycle. Rather, changes in SLBP abundance are
regulated through post-transcriptional mechanisms. Translation of the mRNA is
up-regulated at the G1/S transition and remains elevated during S-phase.
Following S-phase, SLBP is rapidly degraded through a proteasome-dependent
pathway. Hence, as a result of its controlled synthesis and degradation, the
expression of SLBP is restricted to G1/S and S-phase of the cell cycle
(Whitfield et al., 2000
). The
cell cycle-regulated expression of SLBP is thus a major mechanism through
which accumulation and translation of mRNAs encoding the replication-dependent
histones are restricted to S-phase, although increased histone gene
transcription also occurs at this time
(Eliassen et al., 1998
).
In contrast to somatic cells, accumulation and translation of
replication-dependent histone mRNAs are not linked to S-phase in oocytes and
embryos. Oogonia undergo a final S-phase during embryonic development of the
female, then enter meiosis (and are termed oocytes) and a prolonged period of
G2-arrest that may last for many years. Shortly before ovulation and
fertilization, oocytes undergo meiotic maturation, during which they are
released from G2-arrest, enter M-phase, and complete the first meiotic
division before becoming arrested at metaphase II (reviewed by
Wassarman, 1988). Following
fertilization, embryos embark on a series of cleavage divisions consisting of
alternating periods of DNA replication and mitosis with no increase in cell
mass. In mammals, unlike frogs and flies, these early cycles are slow (15-20
hours) and include clearly defined G1 and G2 phases
(Bolton et al., 1984
;
Howlett and Bolton, 1985
). In
the mouse, replication-dependent histone mRNAs and proteins are present in
oocytes and embryos (Wassarman and Mrozak,
1981
; Graves et al.,
1985
; Clarke et al.,
1997
; Wiekowski et al.,
1997
), and translation of the mRNAs is not coupled to DNA
replication in early embryos (Wiekowski et
al., 1997
). These histones are required to replace the protamines
of the sperm DNA and to assemble newly synthesized embryonic DNA into
chromatin until the embryo becomes transcriptionally active.
To investigate the mechanism underlying this pattern of histone expression, we tested the hypothesis that SLBP expression and activity were not restricted to S-phase in oocytes and early embryos. We report that, in contrast to proliferating somatic cells, SLBP is expressed and possesses stem-loop binding activity both in oocytes and in embryos at all stages of the cell cycle. Thus, SLBP expression is uncoupled from progression through S-phase in the oocyte and early embryo. These results suggest that the factors regulating SLBP expression and activity may be distinct from those regulating other aspects of cell cycle progression. In addition, they provide a potential molecular explanation for the distinctive pattern of histone gene expression in oocytes and embryos, and they open the possibility that SLBP may perform additional roles in these cell types.
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Materials and Methods |
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To obtain embryos at different stages of preimplantation development, CD-1
females (8 weeks or older, Charles River Canada) were injected with 7.5 IU of
pregnant mares' serum gonadotropin followed 44-48 hours later by 5 IU of human
chorionic gonadotropin (hCG) and caged individually with males overnight. For
early and late 1-cell embryos and early 2-cell embryos, plugged females were
sacrificed at 16 hours post-hCG, and the 1-cell embryos were recovered and
cultured until they reached the appropriate stage. For late 2-cell and older
embryos, plugged females were sacrificed at 42 hours post-hCG, and the 2-cell
embryos were flushed from the oviduct and cultured until they reached the
appropriate stage. Embryo stages were obtained at the following times
post-hCG, as previously described (Bolton
et al., 1984; Howlett and
Bolton, 1985
; Smith and
Johnson, 1986
): early 1-cell (G1), 18 hours; mid 1-cell (S), 24
hours; late 1-cell (G2), 28 hours; early 2-cell (G1/S), 32 hours; late 2-cell
(G2) 44 hours; early 4-cell (G1/S) 52 hours; late 4-cell (G2), 64 hours.
Blastocysts were collected at 116 hours post-hCG. Embryos were cultured in 5
µl drops of KSOM (Lawitts and Biggers,
1993
) under paraffin oil at 37°C in an atmosphere of 5%
CO2 in air.
Immunoblotting
Oocytes or embryos were collected in 5 µl of loading buffer
(Harlow and Lane, 1988) and
frozen at -80°C until use. Samples were electrophoresed through 10% or 12%
polyacrylamide gels in Tris glycine buffer (25 mM Tris, 250 mM glycine, 0.1%
SDS, pH 8.3). Proteins were transferred onto a PVDF membrane (Amersham) for
120-150 minutes at 70 V in a transfer buffer containing 25 mM Tris, 192 mM
glycine, 20% v/v methanol, pH 8.3. Membranes were soaked for 30 minutes in
blocking solution (5% non-fat milk in TBS), and then incubated with gentle
agitation in anti-SLBP antiserum (Wang et
al., 1996
; Whitfield et al.,
2000
) diluted 1:4000 in blocking buffer at 4°C overnight.
Following three washes in TBS containing 0.1% Tween-20 (TBST), membranes were
incubated for 1 hour in biotinylated anti-rabbit IgG (Jackson Immunoresearch)
diluted 1:5000 in TBST, washed as above, incubated for 30 minutes in
streptavidin-horse radish peroxidase (Amersham) diluted 1:1000 in TBST, and
washed as above. Immunoreactive proteins were revealed using ECL Plus
(Amersham) according to the manufacturer's instructions.
Immunofluorescence
Oocytes and embryos were freed of the zona pellucida using acidified (pH
2.5) Tyrode's solution, washed in PBS, and fixed for 15 minutes at room
temperature in a freshly prepared solution of 4% paraformaldehyde in PBS. The
fixed cells were stored in a blocking solution of PBS, 3% BSA, 0.5% Triton
X-100 at 4°C for up to one week. To detect SLBP, oocytes or embryos were
transferred to affinity-purified anti-SLBP diluted 1:1000 in blocking solution
and incubated with agitation overnight at 4°C. They were then rinsed twice
for 15 minutes in blocking solution, incubated for 1 hour at room temperature
in FITC-conjugated donkey anti-rabbit IgG (Jackson Laboratories) diluted 1:100
in blocking solution, washed twice as previously described and mounted on a
microscope slide in a drop of Moviol (Hoechst) containing 1 µg/ml of DAPI
to stain the DNA and 2.5% (w/v) 1,4-diazabicyclo[2.2.2]octane (Sigma) to
retard extinction of the fluorescent signal. The cells were examined using an
Olympus BX60 microscope equipped for epifluorescence with fluorescein and UV
filter sets and linked to an Applied Imaging analysis system.
Reverse transcription (RT) and polymerase chain reaction (PCR)
RNA was purified, reverse-transcribed, and the cDNA amplified as previously
described (Mohamed et al.,
2001). For each experiment, RNA was prepared from 25-50 oocytes or
embryos and cDNA from 8 oocyte/embryo-equivalents was used in the PCR
reaction. Each PCR cycle (40 cycles) consisted of 1 minute at 94°C, 30
seconds at 57°C and 45 seconds at 72°C. Primers and positions on the
published mRNA sequence (GenBank locus NM_009193) were
81-GGTTATGGGAGTCGCCGCGA-100 and 880-TAACTCATGGCAGAGAAGTC-861. These primers
should generate an 800-nt fragment from cDNA, but are separated by 1.14 kb in
genomic DNA (GenBank locus AC079504). In all experiments, samples prepared
without reverse-transcriptase were included to ensure that the PCR product was
cDNA-dependent. Ten microliters (one-fifth) of amplified product was
electrophoresed through an 8% (w/v) polyacrylamide gel prepared in 45 mM
Tris-borate, 1 mM EDTA, pH 8.0, stained using ethidium bromide, and
photographed using a UV transilluminator.
Phosphatase treatment
Oocytes or embryos were transferred to a microfuge tube in a minimal volume
of medium and stored at -80°C. After thawing, each tube received 5 µl
of phosphatase buffer (Boehringer) containing protease inhibitors (Boehringer)
and 1 µl of phosphatase (Boehringer). The tubes were incubated for 15
minutes at 37°C, after which 5 µl of 2x Laemmli buffer was added.
The samples were processed for immunoblotting as described above.
Mobility-shift assays
Fifty oocytes or embryos collected at MII, G1, S or G2 of the first cell
cycle, or at the blastocyst stage were lysed in 0.1% NP-40, 50 mM Tris-Hcl [pH
7.5], 150 mM NaCl in a final volume of 25 µl and stored at -80°C until
use. Five hundred oocytes collected from 15-day-old mice were collected and
stored in 25 µl of the same buffer. An appropriate volume of each extract
was mixed on ice with 0.5 pmol of end-labeled, 30 nt stem-loop RNA either in
the presence or absence of 1 µg of protein A-purified SLBP antibody in a
total volume of 20 µl. Each reaction also contained 10 µg tRNA as a
competitor for non-specific RNA binding activities, 20 mM EDTA, 10 µg BSA
and 5 µl Buffer D (20 mM HEPES-KOH, [pH 7.9], 100 mM KCl, 0.2 mM EDTA [pH
8.0], 20% glycerol). Following a 10 minute incubation, complexes were resolved
on a 7% native polyacrylamide gel (37.5 parts acrylamide to 1 part
bisacrylamide) containing Tris-borate-EDTA buffer. The gel was dried and
subject to autoradiography. The preparation and labeling of the stem-loop RNA
and protein A-purification of the SLBP antibody have been described previously
(Dominski et al., 1999;
Wang et al., 1996
).
Drugs
Puromycin (Sigma) was dissolved in water at 10 mg/ml and used at 10
µg/ml. Nocodazole (Sigma) was dissolved in water at 150 µg/ml and used
at 0.15 µg/ml. For each experiment, the effectiveness of the drugs was
confirmed by their ability to prevent cleavage. Phosphatase (Boehringer) was
stored according to the manufacturer's instructions. Roscovitine (Sigma) was
dissolved in DMSO at 10 mM and used at 100 µM. For each experiment, its
effectiveness was confirmed by its ability to prevent germinal vesicle
breakdown (GVBD) in a sample of oocytes. U0126 (Calbiochem) was dissolved in
DMSO at 10 mM and used at 0.5 µM. For each experiment, its effectiveness
was confirmed by immunoblotting using an anti-phospho-ERK antibody (Santa
Cruz).
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Results |
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Upon hormonal stimulation of the somatic follicular cells, fully grown
oocytes undergo meiotic maturation. During maturation, the oocyte undergoes
germinal vesicle breakdown (GVBD), enters M-phase of the cell cycle, completes
the first meiotic division, and becomes arrested at metaphase of meiosis II.
Oocytes at metaphase II contained substantially more SLBP than oocytes at
prophase I, as determined both by immunoblotting
(Fig. 1A, lane 2) and
immunofluorescence (Fig. 1B,
lower panel). SLBP in M-phase oocytes was uniformly distributed throughout the
egg. In contrast to the increase in SLBP protein during maturation, the
quantity of SLBP mRNA did not detectably increase as evaluated by RT-PCR
(Fig. 1C), consistent with the
general transcriptional arrest during oocyte maturation
(Masui and Clarke, 1979;
Wassarman, 1988
). SLBP protein
quantity did not increase when prophase I oocytes were incubated for up to 12
hours in the presence of the proteasomal inhibitor, MG132, implying that the
increase during maturation is not due to reduced proteasome-dependent
degradation (data not shown).
To determine the timing of SLBP accumulation during meiotic maturation, we took advantage of the fact that when GV-stage oocytes are removed from ovarian follicles and placed in culture, they undergo maturation to metaphase II. During maturation in vitro, GVBD has occurred by 2 hours of incubation and the first polar body has been expelled by 9-12 hours. Immature oocytes were collected, placed in culture, and samples were collected at 3-hour intervals. Only oocytes that had undergone GVBD were collected at each time point (except the 0-hour sample which contained GV-stage oocytes). By 3 hours of incubation, a significant increase in the amount of SLBP was detectable (Fig. 2, lanes 1,2), and the SLBP showed a slower mobility suggestive of phosphorylation (see below). As maturation continued, the amount of SLBP progressively increased. When oocytes that had reached metaphase II, as indicated by the presence of the first polar body (Fig. 2, lane 5), were incubated for an additional 3 or 15 hours, the quantity of SLBP continued to increase (Fig. 2, lanes 6,7). Thus, by shortly after GVBD, SLBP has begun to accumulate in maturing oocytes and it continues to accumulate even after the oocytes have reached metaphase II.
|
To determine whether the high level of SLBP in mature oocytes was
maintained during early embryonic development, embryos at different stages of
preimplantation development were collected and analyzed by immunoblotting.
SLBP remained abundant throughout the first cell cycle and the early portion
of the second cell cycle (Fig.
3A, lanes 2,3). Notably, there was no reduction in SLBP level at
the end of the first S-phase or during mitosis to the 2-cell stage, in
contrast to its degradation at the end of S-phase in somatic cells
(Whitfield et al., 2000).
However, SLBP levels had decreased significantly by the late 2-cell stage
(Fig. 3A, lane 4). A further
decrease was evident between the late 2-cell stage and the early 4-cell stage,
and SLBP declined to a very low level by the late 4-cell stage
(Fig. 3A, lanes 5,6). SLBP
remained present at a low level throughout subsequent preimplantation
development, as exemplified by the signal in the lane containing 100 embryos
(about 3200 cells) at the blastocyst stage
(Fig. 3A, lane 7). SLBP mRNA
was present throughout preimplantation development, but the amount of RT-PCR
product declined after the 2-cell stage
(Fig. 3B) which is consistent
with the widespread loss of maternal mRNA that occurs at this time
(Paynton et al., 1988
), and
increased again by the blastocyst stage presumably as a result of the
increased cell number. Thus, the quantity of SLBP protein remains high during
the first two cell cycles, then declines to a much lower level once the embryo
reaches the 4-cell stage and remains low during subsequent development.
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To determine whether on-going protein synthesis was required to maintain the high level of SLBP in early embryos, 1- and 2-cell embryos were treated with the protein synthesis inhibitor, puromycin. Embryos treated for either 6 or 12 hours contained less SLBP than control embryos processed at the same time, although a substantial fraction of SLBP remained (Fig. 3C, compare lanes 2 vs 3, 5 vs 6, and 8 vs 9). This implies that SLBP, which has accumulated during meiotic maturation, continues to be synthesized during the first two cell cycles.
As revealed by immunofluorescence, SLBP was present in the nucleus and cytoplasm during the early embryonic cell cycles, including both the male and the female pronucleus at the 1-cell stage (Fig. 4). Some SLBP was also present in the second polar body (Fig. 4A), and remained there as the embryo developed (Fig. 4D2). The intensity of staining was consistently greater in the nucleus suggesting that, as in oocytes, SLBP preferentially accumulates in this compartment. Embryos fixed at G1, S, and G2 of the first three cell cycles showed no obvious differences in the nucleo-cytoplasmic distribution of SLBP (data not shown). However, embryos fixed early in a cell cycle occasionally contained very small nuclei, which presumably were at late telophase following a recent mitosis. SLBP was not detectable in these nuclei, although it was present in the surrounding cytoplasm (Fig. 4C). This suggests that SLBP accumulates in the nuclei following each mitosis. In embryos beyond the 4-cell stage, SLBP staining was only weakly detected by immunofluorescence (Fig. 4D), consistent with the relatively low quantity detected by immunoblotting.
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SLBP is the sole stem-loop binding activity in oocytes and
embryos
As SLBP exerts its biological function through binding to the stem-loop
region of mRNAs encoding histones, we tested whether the SLBP in oocytes and
embryos was active in RNA binding and whether there were other factors that
could bind the 3'-end of the histone mRNA. To this end, we employed a
mobility-shift assay in which cellular lysates are mixed with a radiolabelled
histone mRNA stem-loop and then fractionated by electrophoresis to determine
whether migration of the labelled stem-loop is retarded
(Williams and Marzluff, 1995;
Wang et al., 1996
). Both
GV-stage and metaphase II oocytes contained an activity able to bind the
stem-loop sequence. Ten metaphase oocytes contained more binding activity than
over 200 GV-stage oocytes (Fig.
5A, lanes 2,4), which is consistent with the accumulation of SLBP
during oocyte maturation as analyzed by Western blotting
(Fig. 1A;
Fig. 2A). Furthermore, all of
the binding activity in both GV and metaphase oocytes was supershifted by
addition of the anti-SLBP antibody (Fig.
5A, lanes 3,5). This strongly suggests that the single SLBP
species recognized by the antibody accounts for all of the detectable
stem-loop binding activity in oocytes.
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The same assay was used to evaluate the stem-loop binding activity in embryo lysates. Stem-loop binding activity was present in 1-cell embryos at G1, S, and G2 of the cell cycle (Fig. 5B, lanes 4,6,8). There was no substantial difference in the binding activity of metaphase II oocytes and the G1-phase embryos (Fig. 5B, compare lanes 2 and 4). This suggests that the phosphorylation of SLBP at M-phase does not affect its ability to bind the histone mRNA stem-loop in vitro. The doublet observed in the complexes from metaphase II samples (lane 2) is a result of the phosphorylation of SLBP (J.A.E. and W.F.M., unpublished; see below). The amount of SLBP binding activity increased, however, as embryos progressed through the first cell cycle (compare lanes 4, 6 and 8), even though the amount of SLBP remained essentially unchanged as analyzed by Western blotting (Fig. 5C). Extracts prepared from early and late blastocyst embryos also contained stem-loop binding activity (Fig. 5D, lanes 3,4). The amount of activity was considerably lower than in metaphase II oocytes (compare lane 2 vs 3 and 4), in agreement with the reduced amount of SLBP detected by Western blotting at these stages (Fig. 3). As in the oocytes, all detectable stem-loop binding activity in embryos was supershifted by the addition of the anti-SLBP antibody (Fig. 5B, lanes 3,5,7,9), demonstrating that both oocytes and embryos contain a single stem-loop binding activity.
SLBP is phosphorylated at meiotic and mitotic M-phase
As shown in Fig. 2, the
electrophoretic mobility of SLBP decreased during meiotic maturation. This
mobility shift was evident shortly after GVBD and remained throughout
maturation to metaphase II. To test whether phosphorylation contributed to the
decrease in electrophoretic mobility, metaphase oocyte lysates were treated
with phosphatase prior to immunoblotting. Phosphatase treatment converted SLBP
to the fast-migrating form observed in GV-stage oocytes
(Fig. 6A), indicating that SLBP
becomes phosphorylated during meiotic maturation.
|
To test whether phosphorylation of SLBP was reversed when activated oocytes exited M-phase, metaphase II oocytes were exposed to SrCl2 to induce parthenogenetic activation and samples were collected at different times after exposure to SrCl2. By 2 hour post-activation, SLBP had been converted to the fast-migrating dephosphorylated form (Fig. 6B). This form remained predominant at 4 hour and 6 hour post-activation. Conversion of SLBP to the fast-migrating form following activation is also evident in Fig. 3A (compare lanes 1 and 2). Thus, the phosphorylation of SLBP upon entry into M-phase of meiosis is reversed upon activation and exit from M-phase.
The rapid phosphorylation of SLBP following GVBD and its rapid
dephosphorylation following activation suggested that phosphorylation might
depend on cyclin-dependent kinase 1 (cdk-1) activity, which increases rapidly
at GVBD and remains high throughout maturation and in oocytes arrested in
metaphase II, and falls rapidly after completion of meiosis
(Choi et al., 1991). To test
this, oocytes at different stages of maturation were treated with roscovitine,
an inhibitor of cdk activities (Meijer et
al., 1997
; Deng and Shen,
2001
). When oocytes were treated at prophase I, they failed to
undergo germinal vesicle breakdown and SLBP remained in its fast-migrating,
hypophosphorylated form (Fig.
6C, lanes 1-3). Moreover, SLBP did not accumulate
(Fig. 6C, lane 3), suggesting
that accumulation of SLBP also depends on cdk1 kinase activity. When oocytes
that had undergone GVBD and contained slow-migrating SLBP were treated with
roscovitine for 6 hours, SLBP was converted to the fast-migrating form by the
end of the treatment period (lanes 4-6). Following transfer of oocytes to
roscovitine-free medium, SLBP was converted back to the slow-migrating form
(data not shown), indicating that the effect of the drug was reversible. In
contrast, exposure of oocytes to an inhibitor of the ERK MAP kinases, U0126,
did not affect the electrophoretic mobility of SLBP (lane 7). Similarly, when
oocytes that had reached metaphase II were treated with roscovitine, the
fast-migrating form of SLBP was predominant
(Fig. 6C, lane 9), whereas the
slow-migrating form was predominant following treatment with U0126
(Fig. 6C, lane 10). These
results indicate that cdk1 activity is continuously necessary during
maturation to maintain the bulk of SLBP in its slow-migrating phosphorylated
form, suggesting that it can be rapidly dephosphorylated during this time.
Since SLBP becomes phosphorylated by a cdk-dependent pathway in maturing oocytes, we then examined whether SLBP also becomes phosphorylated at mitotic M-phase of early embryos, when there also is high cdk1 activity. One- and 2-cell embryos were transferred at times corresponding to late G2 of each cell cycle to medium either containing nocodazole to arrest them at M-phase, or containing puromycin to prevent the transition from G2 to M-phase, or were left untreated. Six or 12 hours later, when the untreated controls had undergone cleavage, all groups were collected for immunoblotting. As shown in Fig. 6D, SLBP in both 1- and 2-cell embryos treated with nocodazole displayed the retarded electrophoretic mobility indicating it was phosphorylated (lanes 3,7). This change in mobility was not observed in the puromycin-treated embryos (lanes 2,6), however, indicating that the presence of the slow-migrating SLBP in the nocodazole-treated embryos was not a non-specific consequence of arresting cell-cycle progression. In addition, puromycin treatment at late G2 of the second cell cycle prevented the rapid decrease in SLBP (Fig. 6D), lane 6), while treatment with nocodazole did not (Fig. 6D, lane 7), consistent with a degradation of SLBP at late G2 or entry into mitosis of the second cell cycle. Thus, SLBP also becomes phosphorylated at mitotic M-phase of the early embryonic cell cycles, probably by a cdk1-dependent pathway, even though SLBP remains stable during this time.
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Discussion |
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These results demonstrate that the cell-cycle dynamics of SLBP synthesis
and degradation differ markedly in oocytes and early embryos as compared to
somatic cells. First, in somatic cells, SLBP becomes phosphorylated at the end
of S-phase (Whitfield et al.,
2000), probably by cdk2, and this targets it for
proteasome-dependent degradation (L.X. Zheng and W.F.M., unpublished). Thus,
no SLBP is detected in G2 cells (Whitfield
et al., 2000
). In contrast, the presence of SLBP in G2-arrested
oocytes, together with the inability of a proteasome inhibitor to increase
SLBP quantity at this stage, implies that the mechanism that degrades SLBP
following S-phase in somatic cells is not active in oocytes.
Second, the substantial accumulation of SLBP during M-phase of meiotic
maturation indicates that it is abundantly synthesized at this stage of the
cell cycle, whereas this is not the case in somatic cells. In the case of
other proteins that accumulate during oocyte maturation, including tissue-type
plasminogen activator [tPA (Huarte et al.,
1987)], HPRT (Paynton et al.,
1988
), mos (O'Keefe et al.,
1989
; Paules et al.,
1989
; Gebauer et al.,
1994
), FGF receptor (Culp and
Musci, 1999
) cyclin B
(Polanski et al., 1998
;
Barkoff et al., 2000
;
Tay et al., 2000
) and spindlin
(Oh et al., 2000
), this is due
to increased translation of existing mRNAs. Translation of these mRNAs is
regulated by U-rich sequences, termed adenylation control elements (ACE) or
cytoplasmic polyadenylation elements (CPE), that are located in the
3'-untranslated region (utr) of the mRNA within about 100 nt of the
polyadenylation signal (Richter,
1995
; Gray and Wickens,
1998
; Oh et al.,
2000
). The ACE likely also represses translation of these mRNAs in
immature oocytes (Stutz et al.,
1997
; Stutz et al.,
1998
). The increase in SLBP synthesis in M-phase oocytes may
depend on interaction of oocyte factors with analogous elements in the
3'-utr of SLBP mRNA.
Third, our results indicate that, in early embryonic cells, the quantity of
SLBP depends primarily on progression through development or on time elapsed
since fertilization, rather than on cell cycle stage. The lifespan of most of
the oocyte proteins inherited by the embryo is not known. However, most
maternal mRNA is degraded by the 2-cell stage
(Paynton et al., 1988) and we
observed a decrease in the amount of RT-PCR product detected at this stage.
Hence, a loss of maternal SLBP mRNA may be one factor contributing to the drop
in SLBP levels near the end of the second cell cycle. Indeed, as indicated by
the reduced quantity present in 1- and 2-cell embryos exposed to puromycin,
SLBP is both synthesized and degraded following fertilization. These
considerations suggest that the large amount of SLBP present during the early
embryonic cell cycles represents maternal inheritance, both of the protein and
of the mRNA that is translated after fertilization. This maternal supply
becomes depleted near the end of the second cell cycle and during the third
cell cycle, and the much smaller quantity of SLBP detectable in late 4-cell
embryos and blastocysts is the product of embryonic gene activity. Thus, SLBP
may become appropriately cell cycle-regulated, with protein expression
restricted to S-phase, beginning at the fourth cell cycle.
The persistence of SLBP throughout the cell cycle allowed us to establish
that it underwent an M-phase-specific phsophorylation that was reversed upon
exit from M-phase. These results do not exclude that SLBP may also be
phosphorylated, perhaps at different sites, at other phases of the cell cycle.
M-phase phosphorylation is sensitive to roscovitine, implying that it requires
the activity of cyclin-dependent kinases. The timing of SLBP phosphorylation
during M-phase, as well as its rapid dephosphorylation following egg
activation, closely matches the activity of cdk1 [cdc2
(Choi et al., 1991)], which
suggests that this is likely the regulatory kinase. As roscovitine treatment
of oocytes at midor late maturation led to dephosphorylation of much of the
SLBP, it appears that on-going cdk1 activity during maturation is required to
maintain the bulk of SLBP in its M-phase phosphorylated form.
SLBP in oocytes and at all stages of the first cell cycle was able to bind RNA containing the histone stem-loop in vitro. These results indicate that the M-phase phosphorylation of SLBP did not prevent its stem-loop binding activity. They also reveal that the stem-loop binding activity increased as cells progressed through the first cell cycle, even though the quantity of SLBP protein did not. This could mean that SLBP stem-loop binding is enhanced by co-factors whose activity increases during progression through the cell cycle, or that some of the maternal histone mRNA becomes degraded during this period of time, thus releasing SLBP that can bind to the labeled probe in vitro. It will be important to establish whether SLBP at G1, G2 and M-phase of the cell cycle is able to promote processing and translation of stem-loop mRNAs.
SLBP and histone gene expression in oocytes and embryos
Histone gene expression in mammalian oocytes and early embryos differs in
several respects from proliferating somatic cells. For example,
replication-dependent histone mRNAs are stable and accumulate in oocytes
arrested at G2 (Wassarman and Mrozak,
1981; Graves et al.,
1985
; Clarke et al.,
1997
), whereas they are degraded at G2 in somatic cells. The
distinctive expression of SLBP in oocytes and embryos suggests a potential
mechanistic basis for these differences. As SLBP stabilizes histone mRNA by
stimulating 3'-processing of the primary transcripts and remains
associated with the histone mRNP, it is likely that the expression of SLBP in
oocytes enables these cells to store replication-dependent histone mRNAs
during G2 arrest.
In addition, the increase in SLBP during maturation immediately precedes
the increased efficiency of histone synthesis in fertilized mouse eggs
(Schultz, 1986;
Wiekowski et al., 1997
). SLBP
is required for the translation of stem-loop histone mRNAs
(Sun et al., 1992
;
Gallie et al., 1996
), and we
have recently shown that SLBP stimulates translation of histone mRNA in vivo
and in vitro (Sanchez and Marzluff,
2002
). The SLBP that accumulates during maturation may play a role
in activating translation of histone mRNA during oocyte maturation and after
fertilization, thus providing the histone proteins necessary both for
repackaging the sperm chromatin and for synthesis of histone proteins for the
first S-phase. The fact that SLBP in oocytes and embryos at all stages of the
cell cycle can bind the histone mRNA stem-loop is consistent with such a
function.
SLBP in oocytes and embryos of other species
SLBP has also been identified in oocytes and early embryos of
Xenopus (Wang et al.,
1999), Drosophila
(Sullivan et al., 2001
) and
C. elegans (Kodama et al.,
2002
; Pettitt et al.,
2002
). However, cell cycle-dependent changes in expression and
activity have been examined only in Xenopus oocytes and not in
embryos of any of these species. Xenopus oocytes express two SLBP
species (Wang et al., 1999
).
xSLBP1 is similar in amino acid sequence to the SLBP identified in mouse and
human. It is present at high levels in G2-arrested growing oocytes but, unlike
mouse SLBP, does not accumulate substantially upon entry into M-phase during
meiotic maturation. xSLBP1 persists at a high level during early
embryogenesis, but its expression during these cell cycles, which differ from
mammalian cell cycles in that they lack gap phases, has not been examined.
xSLBP2 is encoded by a separate gene and is similar to xSLBP1 only in the RNA
binding domain. It also is present in G2-arrested growing oocytes, but is
degraded at oocyte maturation. Histone mRNAs are mainly bound to xSLBP2 during
oocyte growth, but exchange this for xSLBP1 during meiotic maturation
(Wang et al., 1999
). xSLBP2,
although able to bind to the histone mRNA stem-loop, does not support pre-mRNA
processing (Ingledue et al.,
2000
). It is thought be necessary for storage of the large
quantity of translationally silent histone mRNAs that accumulate during oocyte
growth (Wang et al.,
1999
).
Our results demonstrate that, in contrast to Xenopus, mouse
oocytes and embryos contain only a single SLBP species. First, the amount of
stem-loop binding activity, as measured by the band-shift assay, changed in
parallel with the quantity of SLBP, as measured by immunoblotting, at
different developmental stages. Second, at all developmental stages, all of
the in vitro-binding activity was supershifted by addition of the anti-SLBP.
These results imply that the cells do not contain an SLBP2-like protein.
Similarly, only one SLBP is present in oocytes of Drosophila
(Sullivan et al., 2001) and in
the genome of Drosophila
(Sullivan et al., 2001
) and
C. elegans (Martin et al.,
1997
), and a search of the human genome did not reveal any other
genes with similarity to human SLBP in the RNA-binding domain. This difference
between mouse and Xenopus may reflect the much greater accumulation
of histone mRNAs during amphibian oogenesis and the unique mechanism for
storing them in translationally inactive form.
Finally, it should be noted that the amount of SLBP in the oocyte and
1-cell embryo is much higher than the amount of SLBP in the blastocyst embryo,
even though the levels of histone mRNA are similar at these two stages
(Giebelhaus et al., 1983;
Graves et al., 1985
). There
thus appears to be a large excess of SLBP in the mature oocyte and early
embryo, compared with the need for SLBP for synthesis and translation of
histone mRNA. This taken together with the expression of SLBP throughout the
cell cycle open the possibility that SLBP may perform other functions not
directly linked to histone mRNA metabolism
(Abbott et al., 1999
).
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Acknowledgments |
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Footnotes |
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References |
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