|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online November 7, 2008
doi: 10.1242/10.1242/dev.023747
,RIKEN Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Kobe 650-0047, Japan.
Authors for correspondence (e-mail:
bthuy{at}konkuk.ac.kr
and
teru{at}cdb.riken.jp)
Accepted 23 September 2008
 |
SUMMARY |
|---|
 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key words: Nuclear transfer, Chromatin remodeling, Histone acetylation, Histone methylation, Oocyte cytoplasm
|
INTRODUCTION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
;
Wilmut et al., 2002). To date,
almost all animals cloned from somatic cell nuclei have been produced using
enucleated meiosis stage II (MII) oocytes as recipient cytoplasts
(Greda et al., 2006). However,
enucleated germinal vesicle stage (GV) oocytes, which are thought to be better
recipients than MII oocytes (Bao et al.,
2000), or enucleated zygotes have failed to support cloned embryo
development to term (McGrath and Solter,
1984). We previously reported that the use of zygotes as
recipients damaged the transferred donor nuclei and concluded that there was
no firm reason for using this approach for mouse cloning
(McGrath and Solter, 1984;
Wakayama et al., 2000). The
cytoplasm of GV oocytes and zygotes was therefore though to be unsuitable for
nuclear transfer. However, Greda et al. and Egli et al. then suggested that
one or more factors crucial for embryonic development or reprogramming might
be localized to the pronuclei during interphase and that enucleation must
remove them (Egli et al.,
2007; Greda et al.,
2006). They developed alternative methods of enucleation, which
left the reprogramming factors in the zygote cytoplasm. They removed chromatin
either by rupturing the pronuclear envelope or by extracting the chromosomes
during the first zygotic mitosis. They showed support for this by generating
cloned mice from eight-cell blastomeres and from embryonic stem (ES) cells
using enucleated zygotes as recipients. However, the GV oocyte is though to be
a superior nuclear recipient for cloning. In the mouse, immature oocytes can
reprogram the immature nucleus, whereas MII oocytes cannot
(Bao et al., 2000;
Obata et al., 2002). Moreover,
passing the donor nucleus through the cytoplasm of the oocyte at these earlier
stages of oogenesis might further improve reprogramming
(Obata et al., 2002). Yet no
cloned mice have been produced using GV oocytes. Gao et al.
(Gao et al., 2002) concluded
that when the GV is removed before nuclear envelope breakdown and meiotic
metaphase arrest, the oocytes become unsuitable as recipients for nuclear
transfer, leading to abnormal cell division
(Polanski et al., 2005).
Moreover, Ogushi et al. (Ogushi et al.,
2008) report that the nucleolus of the GV stage nucleus is not
necessary for oocyte maturation but is essential for further embryonic
development. Therefore, crucial nuclear factors must be extracted in the
karyoplasts from GV oocytes or zygotes during enucleation, so that the
remaining cytoplasm can no longer support cloned embryo development.
Reprogramming factors in embryonic stem (ES) cells
(Tada et al., 2001;
Cowan et al., 2005) might
reside in the nucleus, because enucleated cytoplasts generated from ES cells
have failed to reprogram somatic cells (Do
and Scholer, 2004). Here, we reinvestigate whether genomic reprogramming factors are located in the nucleus or cytoplasm of GV oocytes and whether they could improve the reprogramming of somatic cell nuclei. We have attempted to produce cloned mice from differentiated adult somatic cells treated with lysates of GV oocyte cytoplasm, because the production of live offspring is clear evidence of nuclear reprogramming.
|
MATERIALS AND METHODS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
)
containing 100 µg/ml dibutyryl cyclic AMP (dbcAMP) (Sigma-Aldrich, St
Louis, MO, USA). The oocytes were stored in mKSOM medium (Specialty Media,
Phillipsburg, NJ, USA) containing 100 µg/ml dbcAMP at 37°C under 5%
CO2 in air until use.
HDAC activity assay
HDAC activity was measured using the CycLex HDAC Deacetylase Fluorometric
Assay Kit (#CY-1150; Cyclex, Nagano, Japan). Each sample containing 150 nuclei
or 150 enucleated or 150 intact oocytes was transferred into 3 µl of assay
buffer and frozen at -80°C. After thawing, each sample was put into 2
µl of lysis buffer, vortexed for 10 seconds and kept on ice for 20 minutes.
Samples were moved to each well of 96-well microplates and incubated by mixing
fluorescence-labeled acetylated peptide (substrate), HDAC and lysyl
endopeptidase at 37°C. Fluorescence intensity was measured for 30 to 60
minutes at 1- to 2-minute intervals using a microtiter plate fluorometer. The
rate of reaction was measured at constant reaction velocity. Diluted crude
HDAC from MCF4 cells was used as a positive control, and MCF4 cells treated
with trichostatin A (TSA) were used as negative controls according to the
manufacturer's instructions. The positive control activity was set arbitrarily
to 100% and all other bands were expressed relative to that as a mean
percentage±s.e.m.
Nuclear transfer to GV oocytes and maturation
Isolated GV oocytes were transferred to mFHM medium containing cytochalasin
D (1 µg/ml) for 30 minutes. This produced a concavity in the oocyte surface
to insert an enucleation pipette (inner diameter 25 µm) to remove the GV
nucleus using a piezo-actuated micromanipulator system (Prime Tech, Aburaki,
Tokyo, Japan). Enucleated oocytes were washed and cultured in mKSOM medium
containing 100 µg/ml dbcAMP until injection. Cumulus cells were injected
into enucleated oocytes using injection pipettes (inner diameter 6-8 µm).
Oocytes were then cultured in mKSOM for various times to obtain the following
meiotic stages: germinal vesicle (GV, 0-3 hours); metaphase I (MI, 7 hours);
anaphase-telophase I (AI-TI, 9-11 hours) and MII (17 hours).
Nuclear transfer to MII oocytes and activation
Cumulus cell donor and the recipient oocyte were collected from BDF1
females. Nuclear transfer to MII oocytes, and oocyte activation methods were
as described (Kishigami and Wakayama,
2007; Wakayama,
2007).
Somatic cell permeabilization and preparation of oocyte cytoplasmic lysates
Cumulus cells were washed in Ca2+- and Mg2+-free
Hanks' balanced salt solution (Gibco-BRL, Gaithersburg, MD, USA) and incubated
in 200 ng/ml streptolysin O in Hanks' solution for 40 minutes at 37°C. In
preliminary experiments, 90% of cells were permeabilized, as judged by vital
staining with 0.4% Trypan Blue (Gibco-BRL). Oocyte cytoplasmic lysates were
prepared as follows: 500 GV oocytes were collected and zonae pellucidae were
removed using acidic Tyrode's solution. Zona-free oocytes were broken down in
5 µl droplets of HEPES-buffered medium (HEPES-CZB)
(Chatot et al., 1990)
containing an ATP-generating system (1 mM ATP, 10 mM creatine phosphate, 25
µg/ml creatine kinase, 100 µM GTP) and protease inhibitors
(Sigma-Aldrich). Entire GVs were removed
(Fig. 1B). Removal of all GVs
was confirmed using nuclear staining with 12 µg/ml Hoechst 33342
(Polysciences, Warrington, PA, USA) for 15 minutes. Permeabilized cumulus
cells were incubated in 5 µl droplets of oocyte cytoplasmic lysates for 45
minutes at 37°C with 1000 cells per lysate. Another group of treated
cumulus cells was cultured for 2 hours with RPMI-1640 medium (Gibco-BRL)
containing 10% fetal calf serum (FCS), antibiotics and 2 mM CaCl2
to reseal the plasma membrane (Fig.
1C). Control permeabilized cumulus cells were incubated in the
same medium without oocyte cytoplasmic lysates. Treated cumulus cells were
then used for nuclear transfer.
Reprogramming of fibroblasts by GV cytoplasmic lysate treatment
Mouse fibroblasts collected from the tails of adult mice were cultured in
DMEM medium until used. Cells were collected and separated by trypsinization
and the cell suspensions were permeabilized and treated with GV lysate, and
then resealed membrane as above. Treated cells were cultured in DMEM or ES
medium for 1, 2, 3 or 4 weeks, corresponding to 2, 4, 6 or 8 passages. ES
cells were used as controls. After culture, cells were collected to examine
Oct4 expression and nuclear lamin A (LMNA, a marker of differentiated cells)
repression using RT-PCR and real-time quantitative RT-PCR.
Polymerase chain reaction
mRNA was extracted using the Dynabeads mRNA Direct Kit (Dynal Asa, Oslo,
Norway), according to the manufacturer's instructions and eluted in 15 µl
DEPC-treated water. Reverse transcription (RT) was performed with 8 µl mRNA
using the Superscript First-Strand synthesis System (Invitrogen, Carlsbad, CA,
USA) and oligo(dT)15 primers. Quantitative RT-PCR reactions were performed in
triplicate on a DNA Engine OPTICON4 (MJ Research, San Francisco, CA, USA) with
IQ SYBR Green (Bio-Rad, Hercules, CA, USA). Sequences of the primers used were
as follows: OCT4 primer set (GenBank Accession Number, NM_013633; sense,
5'-GGCGTTCTCTTTGGAAAGGTGTTC-3'; antisense,
5'-CTCGAACCACATCCTTCTCT-3' for a 312 bp product); LMNA primer set
(GenBank Accession Number, NM_001002011; sense,
5'-GCAGGCATGGAGTTCTCTTC-3'; antisense, 5'-CCGTCATTC CCATC
TCTGT-3' for a 253 bp product) and GAPDH primer set (GenBank Accession
Number, NM_008084; sense, 5'-AGGTCGGTGTGAACGGATTTG-3'; antisense,
5'-TGTAGACCATGTAGTTGAGGTCA-3' for a 123 bp product). RT-PCR
conditions were 95°C for 3 minutes and 35
40 cycles of 95°C for 30
seconds, 58°C for 1 minute and 72°C for 1 minute. Quantitative RT-PCR
conditions were 95°C for 10 minutes and 44 cycles of 95°C for 30
seconds, 58°C for 1 minute and 72°C for 1 minute. Data were analyzed
using the 2-ddCt method (Livak and
Schmittgen, 2001) with glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) as a normalization control.
Immunofluorescence
Oocytes and embryos were fixed and then treated as described
(Bui et al., 2004). Primary
antibodies used were: rabbit anti-phospho-histone H3 at serine 10 (P-H3-S10;
Cell Signaling Technology, Danvers, MA, USA); rabbit anti-phospho-histone H3
at serine 28 (P-H3-S28; Upstate Cell Signaling Solutions, Charlottesville, VA,
USA); rabbit anti-trimethylhistone H3 at lysine 9 (Me-H3-K9; Abcam, Cambridge,
UK); mouse anti-
-tubulin or anti-β-tubulin (Sigma-Aldrich) for
staining microtubules; mouse anti-lamin B (Santa Cruz Biotechnology, Santa
Cruz, CA, USA) for nuclear membranes; mouse anti-Cdx2 (BioGenex, San Ramon,
CA, USA) for the trophectoderm (TE) cells; and rabbit anti-Oct4 (H-134, Santa
Cruz Biotechnology) for the blastocyst inner cell mass (ICM). The secondary
antibodies were Alexa-Fluor-568-labeled goat anti-mouse and
Alexa-Fluor-488-labeled chicken anti-rabbit antibodies (Molecular Probes,
Eugene, OR, USA). Control fertilized embryos were produced using
intracytoplasmic sperm injection (ICSI) at the same time.
Quantitative analysis of methylation H3-K9 level in nuclei
Embryos were observed using an Olympus Fluoview FV1000 confocal scanning
laser microscope. Fluorescence intensities of nuclei in pronuclear and
cleavage stage embryos were measured by manually outlining all nuclei in the
display. For blastocysts, at least 15 nuclei were selected from the ICM and TE
cell populations based on morphology (small and compact in the ICM; elongated
in the TE). Nuclei were selected randomly except for metaphase or
anaphase-telophase stages, as shown in strong green in
Fig. 6B, part h, indicating a
typical high level of Me-H3-K9. The total intensity in each nucleus was
measured from five different regions and the background value for the
cytoplasm was subtracted. This was multiplied by the nuclear volume to
represent the total amount of fluorescence for the nucleus.
Statistical analysis
Immunostaining experiments were repeated three times and at least 50
immunostained oocytes were examined in each group. Each HDAC assay was
performed on at least three replicates. The data were normalized by arcsine
transformation for each replication. The transformed values were analyzed
using one-way ANOVA and P<0.05 was considered to be statistically
significant.
|
RESULTS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
), first we injected somatic nuclei into either GV nuclei
or the cytoplasm of GV oocytes (Fig.
1A). Oocytes were then cultured for 2 hours in medium containing
dbcAMP to prevent GVBD and we examined histone H3 acetylation at lysines 9 and
14 (Ac-H3-K9 and Ac-H3-K14). Interestingly, we found that there was a
difference in the level of histone acetylation of somatic chromosomes when
injected into the nucleus versus the oocyte cytoplasm. Ac-H3-K9 and Ac-H3-K14
levels were maintained in somatic chromosomes injected into the oocyte
nucleus, but they decreased in the oocyte cytoplasm after 2 hours (100% of 40
oocytes examined for each case of Ac-H3-K9 and Ac-H3-K14, see
Fig. 2B, parts a-f for
Ac-H3-K14). Thus, the cytoplasm of the GV oocyte contained factors that
reduced histone acetylation in somatic chromosomes. However, these could not
induce histone deacetylation completely, even 7 hours after injection (100% of
45 oocytes examined for each case of Ac-H3-K9 and Ac-H3-K14, see
Fig. 2B, parts g-l for
Ac-H3-K14). In the control nontreated dbcAMP group, there were similar results
for nontreated and treated oocytes after 2 hours; however, both H3-K9 and
H3-K14 become deacetylated in oocyte and somatic chromosomes after GVBD (see
Fig. 2C for Ac-H3-K14).
|
We then tested whether HDAC activities increased after GVBD. To do this, the activity levels of HDACs were measured during oocyte maturation. Oocytes were collected at various times of culture to obtain specific stages of meiosis: 0 hours (GV), 3 hours (GVBD), 7 hours (MI) and 17 hours (MII). Oocytes were then assayed for HDAC activity. HDAC activities did not change significantly during oocyte maturation (Fig. 3B). Finally, to examine whether the cytoplasm alone could induce histone deacetylation, enucleated GV oocytes were injected with cumulus cell nuclei and histone acetylation was examined during oocyte maturation. The histone acetylation level was high in somatic chromosomes immediately after injection (100% at 0 hours for both Ac-H3-K9 and Ac-H3-K14) (see Fig. 3C for Ac-H3-K9) and decreased 3 hours after injection. Histone H3 became deacetylated 7 hours after injection when the somatic chromosomes reached the MI-like stage and by 17 hours for the MII-like stage (100% for both Ac-H3-K9 and Ac-H3-K14) (see Fig. 3C for Ac-H3-K9). Thus, histone H3 can be deacetylated under the effect of the oocyte cytoplasm without the need for oocyte-specific nuclear factors.
|
|
|
) and even in
somatic nuclei injected into MII oocytes
(Bui et al., 2006).
Surprisingly, histone H3 in somatic chromosomes (SC), which was originally
methylated at lysine 9, started to decrease in 90% of injected oocytes after 1
hour (Fig. 4A, part e and
Fig. 4B, part e in SC). This
became demethylated completely 7 hours after injection
(Fig. 4A, part k and 4B, part k
in SC). This was maintained during oocyte maturation (17 hours after nuclear
transfer) (Fig. 5A; see Fig.
S1A in the supplementary material). This histone H3 demethylation pattern was
similar in somatic chromosomes injected into both intact and enucleated
oocytes (Fig. 5A). We tested whether there was any difference in the effect of the GV cytoplasm versus the GV nucleus on the pattern of histone H3 methylation in microinjected somatic nuclei. Somatic nuclei were injected into either the nucleus or the cytoplasm of GV oocytes, and the constructs were cultured in medium containing dbcAMP for 3 hours to examine Me-H3-K9. Histone H3 in the somatic nuclei became demethylated 3 hours after injection into the GV cytoplasm but remained unchanged after injection into the GV nucleus at the same time (Fig. 5B). Thus, GV stage cytoplasm can induce histone demethylation but the GV nucleus cannot.
Phosphorylation of histone H3 at serine 10 (P-H3-S10) and serine 28
(P-H3-S28) are involved in chromosome condensation
(Bui et al., 2004;
Bui et al., 2007). Somatic
nuclei injected into intact or enucleated GV oocytes were examined for levels
of P-H3-S10 and P-H3-S28 to determine the cytoplasmic capacity for chromosome
condensation. Histone H3 was not detected as being phosphorylated at either
serine position in any somatic cell nucleus immediately after injection. It
started to be phosphorylated at S28 and then at S10 1-3 h after injection in
both intact and enucleated GV oocytes, and this was maintained during oocyte
maturation (17 hours) (Fig. 5A;
see Fig. S1B in the supplementary material). Histone H3 phosphorylation
patterns in the SCs were similar to those of the oocyte chromosomes. Thus, the
enucleated GV oocyte cytoplasm retained its capacity for chromosome
condensation following the introduction of a somatic nucleus at interphase.
There were also no differences in somatic chromosome morphology between
enucleated and intact oocytes (Fig.
5A).
|
). After 2-3 hours, the somatic nuclei acquired morphology
similar to the late GV stage in oocyte chromosomes, with individual
filamentous bivalents (Fig. 4B,
part n), as described for GVII-IV in pig oocytes
(Bui et al., 2007). This
morphology is specific for GV oocytes and has never been observed in somatic
cell nuclei. Thus, these somatic chromosome-derived nuclei became remodeled
from the somatic to the GV type (Fig.
5A), even without the GV nucleus. After 7 hours and 17 hours
injection, somatic chromosome can reach to the MI- and MII-like stage,
respectively (Fig. 4B, part j;
see Fig. S1A in the supplementary material). Therefore, the somatic chromosome
showed similar developmental stages of GV oocyte. However, about 10% of donor
nuclei did not change, or remained at the GV stage even 17 hours after
injection (Fig. 5A; see Fig.
S1C in the supplementary material). Although there were no apparent
differences in the nuclear morphology of donor cell nuclei injected into
intact or enucleated GV oocytes (Fig.
5A), when they were observed 17 hours after injection, about 10%
of the enucleated oocytes discharged a giant first polar body and resembled a
two-cell embryo, as reported (Polanski et
al., 2005). However, this was very rare in intact oocytes
(Fig. 5A, asterisk;
Fig. 5C). This was the only
difference between intact and enucleated oocytes, which suggests that
enucleation removed key cell cycle factors from the cytoplasm.
Somatic nuclear histone H3-K9 is demethylated by GV oocyte cytoplasmic lysates
The low efficiency of cloning technology is thought to be caused by
incomplete reprogramming of the donor cells after nuclear transfer. Therefore,
the prevention of epigenetic errors such as DNA hypermethylation has been
expected to lead to an improvement in the success rate in animal cloning
through applying drugs to the donor cells before nuclear transfer
(Enright et al., 2003;
Shi et al., 2003). We found
here that the GV cytoplasm has the potential to remodel somatic cell nuclei.
Next, we tried to reprogram somatic cell nuclei by treatment with GV oocyte
cytoplasmic lysates based on a method for the transdifferentiation of somatic
cells (Hakelien et al., 2002).
Permeabilized cumulus cells were exposed to pure lysates in which the oocyte
nuclei had been removed and were then examined for histone methylation levels
(Fig. 1B). The treated cumulus
cells had low levels of histone H3 methylation
(Fig. 6A, part h) compared with
intact and with permeabilized control cumulus cells
(Fig. 6A, parts b and e). Thus,
the GV oocyte cytoplasmic lysate retains its demethylation activity, even
without the nucleus. We then injected the treated cumulus cells into MII
oocytes matured in vivo. The demethylation of H3-K9 in cumulus cells that had
been treated with GV oocyte cytoplasmic lysates was maintained for 2 hours
after injection into MII oocytes without activation (see SC in Fig. S2B'
in the supplementary material), whereas the cytoplasm of MII oocytes could not
remove the methylation of H3-K9 in intact somatic nuclei (see SC in Fig.
S2A' in the supplementary material). Even after activation, the
pronuclei from cytoplasmic lysate-treated cumulus cells showed low levels of
histone H3-K9 methylation (see SP in Fig. S2D' in the supplementary
material).
|
). We examined the expression of Oct4 and repression of
LMNA in GV cytoplasmic lysate-treated cells after culture for 1, 2, 3 and 4
weeks. Because treatment of fibroblasts with cytoplasmic lysates of mouse or
pig GV oocytes promoted colony formation in medium used for embryonic stem
cell culture (ES medium; H.-T.B., S.W., S.K., J.-H.K., N.V.T. and T.W.,
unpublished, the B6D2F1 fibroblasts were treated with mouse GV cytoplasmic
lysates and cultured in DMEM with 10% FBS (DMEM medium) or in ES medium and
then used for RT-PCR.
|
). Beside that, fully grown GV oocytes regulate
their own micro-environment by oocyte-secreted factors, such as bone
morphogenetic protein 15 or growth differentiation factor 9
(Hussein et al., 2006).
Therefore, the GV cytoplasmic lysate used in this study might have contained
some differentiation factors that entered the treated fibroblasts to induce an
early increase of tissue-specific markers for 1-3 weeks culture
(Fig. 7C).
Embryos reconstructed using somatic cells treated with cytoplasmic lysates from GV oocytes produce high-quality expanded blastocysts
To test whether the changes in histone methylation patterns described above
were associated with nuclear reprogramming, reconstructed oocytes were
activated, the resulting cloned embryos were cultured for 96 h and the quality
of blastocysts was examined. At the pronuclear stage, cloned embryos derived
from lysate-treated cumulus cells showed a lower intensity of methylation than
those derived from intact cumulus cells, similar to control fertilized embryos
in terms of the numbers of asymmetrically methylated female and male pronuclei
(Fig. 6B, parts b,f;
Fig. 6C). The low methylation
level of H3-K9 in the embryos derived from the cumulus cells treated with
cytoplasmic lysates was maintained until the blastocyst stage (96 hours), with
no significant difference from normally fertilized blastocysts
(Fig. 6B, parts h,l;
Fig. 6C). By contrast, cloned
embryos derived from intact cumulus cells showed high methylation levels at
all embryonic stages (Fig. 6B,
parts d,j; Fig. 6C).
Cdx2 and Oct4 are specific gene markers for the TE and ICM, respectively,
at the expanded blastocyst stage (Nichols
et al., 1998; Strumpf et al.,
2005). To assess the quality of blastocysts, TE, ICM and total
cell numbers were evaluated with markers for these genes as described
(Kishigami et al., 2006;
Van Thuan et al., 2006a).
There was no significant difference between groups in the mean numbers of
blastomeres expressing the Cdx2 protein at the blastocyst stage (see Fig. S3
in the supplementary material). However, blastocysts containing more than 15
ICM Oct4-positive blastomeres (designated Type I good embryos)
(Fig. 8A) appeared
significantly more frequently in the lysate-treated group than in the controls
(20% and 7%) (Fig. 8B).
GV cytoplasm enhances somatic cell cloning efficiency
We aimed to produce live offspring by cloning from somatic cell nuclei
using this system, because this endpoint is the strongest evidence for nuclear
reprogramming. As it is difficult to prepare pure GV stage nuclear lysates, we
performed controls in which donor cells were either left intact or
permeabilized and exposed to the same medium lacking cytoplasmic lysates. As
shown in Table 1, the rate of
pronuclear formation and two-cell stage development were no different between
groups. However, when cumulus cells were exposed to GV cytoplasmic lysates and
then transferred into mature enucleated oocytes, we could obtain more live
cloned offspring than in the intact or permeabilized cell group (3.1% from the
cytoplasmic lysate-treated cells versus 0.4% to 0.9% from control groups;
Table 1). These cloned mice
have grown to fertile adulthood (Fig.
8C). Thus, the cytoplasm of the GV stage mouse oocyte can promote
reprogramming of the differentiated somatic cell nucleus without the presence
of the nucleus.
|
|
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
;
Santos et al., 2003).
Previously, we have shown that the methylation level of histone H3-K9 in
somatic nuclei is stable after being injected into MII oocytes in a pig model
(Bui et al., 2006). Here, we
demonstrated for the first time that the GV oocyte cytoplasm, both intact and
enucleated, could demethylate histone H3-K9 in somatic nuclei within 1 hour of
transfer. However, the cytoplasm of MII oocytes appeared to lose this capacity
(see Fig. S2 in the supplementary material). If demethylation of H3-K9 in
somatic cells is one influential reprogramming factor, then our results show
how clones produced from somatic cell nuclei treated with GV oocyte
cytoplasmic lysates can develop to full term.
|
). Methylation of histone H3-K9 remained high during
maturation and activation in pig oocytes
(Bui et al., 2007). The
mechanisms responsible for protecting the maternal chromosomes from histone
demethylation are not clear. Normally, histone methylation is stable
epigenetically and is reversed only during gametogenesis and in early embryos.
However, cloned embryos have higher levels of histone methylation than do
natural embryos. This might result from an incomplete erasure of pre-existing
methylation in the donor cells. It is possible that in somatic cell cloning
with less epigenetic modifications, the lower levels of histone methylation
might be reprogrammed more easily or completely. We found here that the GV
stage cytoplasm induced demethylation of histone H3-K9 in transferred somatic
cell nuclei. We conclude that the GV stage cytoplasm has the ability to
remodel somatic cell nuclei in terms of altering chromatin structure and by
epigenetic modifications.
Extracts of Xenopus oocytes or embryonic cells can remodel
mammalian somatic cell genomes, as shown by the levels of DNA methylation or
gene expression (Byrne et al.,
2003; Simonsson and Gurdon,
2004; Freberg et al.,
2007; Miyamoto et al.,
2007). However, none of these studies managed to produce cloned
offspring, so it is not clear whether these changes involve effective
reprogramming. Our experiments document for the first time that donor nuclei
can be manipulated epigenetically using mammalian GV stage oocyte cytoplasmic
lysates as a step to enhance nuclear reprogramming. We also found that somatic
nuclei could be reprogrammed partially to an embryonic state and express the
pluripotency marker Oct4, and could also dedifferentiate with the repression
of LMNA. The somatic nuclei became demethylated in GV oocyte cytoplasmic
lysates and the resulting embryos showed low levels of histone H3 methylation
after nuclear transfer, similar to normally fertilized embryos. When they
developed to blastocysts, the proportions of cells in the ICM increased
significantly, as indicated by positive staining for Oct4 protein. Thus,
embryos reconstructed from cumulus cell nuclei treated with GV oocyte
cytoplasmic lysates resulted in high-quality blastocysts at a rate about three
times greater than controls. Finally, we obtained live offspring after embryo
transfer with a resulting 3.4-fold increase in the success rate of mouse
cloning. Thus, factors present in the GV cytoplasm, not in the nucleus, can
improve the reprogramming of somatic cell nuclei.
There has been much discussion about the likely effects of enucleation on
cloning efficiency. For example, there is no difference seen in the success
rate of cloning mice between two commonly used methods: enucleation followed
by injection (Wakayama et al.,
1998), versus injection followed by enucleation
(Wakayama et al., 2003). This
suggests that enucleation per se is not the main reason for the low efficiency
of cloning. Here, we confirmed that the reconstructed oocyte retained a large
amount of microtubule-organizing centers and could establish a bipolar spindle
3 hours after nuclear transfer (Fig.
4B, parts f,i), either with or without a GV nucleus, as for the
MII spindle in the oocyte (Van Thuan et
al., 2006b). Histones H3-S10 and H3-S28 of the transferred somatic
cell genomes became phosphorylated and the chromosomes underwent condensation
without the presence of the GV nucleus. This indicated that histone H3 kinase
in the cytoplasm of GV oocytes regulated histone H3 phosphorylation and caused
chromosome condensation (Bui et al.,
2004). Some essential factors are present in the nucleus
(Greda et al., 2006;
Egli et al., 2007;
Ogushi et al., 2008). However,
there must be several reprogramming factors in the cytoplasm at the GV stage.
One possibility is that the nucleus contains cell cycle-related factors rather
than reprogramming factors. We reported that during the process of
spindle-chromosome complex depletion, the main part of 
-tubulin, which
is needed for correct mitotic spindle formation, is removed together with
nuclear mitotic apparatus protein 1 (NuMA1), which is a matrix protein
responsible for spindle pole assembly. These are essential for correct mitotic
spindle formation (Van Thuan et al.,
2006b). Therefore, even though the cytoplasm of enucleated oocytes
might retain some genomic reprogramming factors, the subsequent reconstructed
embryos fail to develop because of abnormal cell division or lack of essential
vital factors. This hypothesis is supported by our observation that abnormal
giant polar bodies were extruded only from enucleated oocytes
(Fig. 5C).
Here, we demonstrate that genomic reprogramming factors in the GV cytoplasm could improve cloning efficiency. We investigated a possible strategy to enhance cloning efficiency through the pre-exposure of somatic cell nuclei to a GV cytoplasmic lysate before microinjection. However, although the mouse is the best model for studying the molecular mechanisms of cloning, using mouse GV cytoplasmic lysates has limitations in practice because we need to kill many animals and it takes a long time to prepare the lysates. However, this study has established the principle of using mammalian GV cytoplasmic lysates for reprogramming of donor somatic cell nuclei. It might be possible to prepare lysates suitable for practical applications using large domestic animals. For example, many porcine GV stage oocytes can be collected quickly by aspiration from ovaries freely available from commercially slaughtered animals. In this study, we aimed to concentrate on the molecular mechanisms of genetic reprogramming involved in cloning technology. Clearly, more studies need to be made on the factors in the GV oocyte cytoplasm that are involved in the remodeling of somatic cell nuclei during cloning by somatic cell nuclear transfer.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/23/3935/DC1
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Present address: School of Biology-Oriented Science and Technology, Kinki
University, Wakayama 649-6493, Japan.
|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Bannister, A. J. and Kouzarides, T. (2005). Reversing histone methylation. Nature 436,1103 -1106.[CrossRef][Medline]
Bao, S., Obata, Y., Carroll, J., Domeki, I. and Kono, T.
(2000). Epigenetic modifications necessary for normal development
are established during oocyte growth in mice. Biol.
Reprod. 62,616
-621.
Bui, H. T., Yamaoka, E. and Miyano, T. (2004).
Involvement of histone H3 (Ser10) phosphorylation in chromosome condensation
without Cdc2 kinase and mitogen-activated protein kinase activation in pig
oocytes. Biol. Reprod.
70,1843
-1851.
Bui, H. T., Van Thuan, N., Wakayama, T. and Miyano, T.
(2006). Chromatin remodeling in somatic cells injected into
mature pig oocytes. Reproduction
131,1037
-1049.
Bui, H. T., Van Thuan, N., Kishigami, S., Wakayama, S., Hikichi,
T., Ohta, H., Mizutani, E., Yamaoka, E., Wakayama, T. and Miyano, T.
(2007). Regulation of chromatin and chromosome morphology by
histone H3 modifications in pig oocytes. Reproduction
133,371
-382.
Byrne, J. A., Simonsson, S., Western, P. S. and Gurdon, J. B. (2003). Nuclei of adult mammalian somatic cells are directly reprogrammed to oct-4 stem cell gene expression by amphibian oocytes. Curr. Biol. 13,1206 -1213.[CrossRef][Medline]
Chatot, C. L., Lewis, J. L., Torres, I. and Ziomek, C. A. (1990). Development of 1-cell embryos from different strains of mice in CZB medium. Biol. Reprod. 42,432 -440.[Abstract]
Cowan, C. A., Atienza, J., Melton, D. A. and Eggan, K.
(2005). Nuclear reprogramming of somatic cells after fusion with
human embryonic stem cells. Science
309,1369
-1373.
Do, J. T. and Scholer, H. R. (2004). Nuclei of
embryonic stem cells reprogram somatic cells. Stem
Cells 22,941
-949.
Egli, D., Rosains, J., Birkhoff, G. and Eggan, K. (2007). Developmental reprogramming after chromosome transfer into mitotic mouse zygotes. Nature 447,679 -685.[CrossRef][Medline]
Enright, B. P., Kubota, C., Yang, X. and Tian, X. C.
(2003). Epigenetic characteristics and development of embryos
cloned from donor cells treated by trichostatin A or 5-aza-2'-deoxycytidine.
Biol. Reprod. 69,896
-901.
Freberg, C. T., Dahl, J. A., Timoskainen, S. and Collas, P.
(2007). Epigenetic reprogramming of OCT4 and NANOG regulatory
regions by embryonal carcinoma cell extract. Mol. Biol.
Cell 18,1543
-1553.
Gao, S., Gasparrini, B., McGarry, M., Ferrier, T., Fletcher, J.,
Harkness, L., De Sousa, P. and Wilmut, I. (2002). Germinal
vesicle material is essential for nucleus remodeling after nuclear transfer.
Biol. Reprod. 67,928
-934.
Greda, P., Karasiewicz, J. and Modlinski, J. A.
(2006). Mouse zygotes as recipients in embryo cloning.
Reproduction 132,741
-748.
Hakelien, A. M., Landsverk, H. B., Robl, J. M., Skalhegg, B. S. and Collas, P. (2002). Reprogramming fibroblasts to express T-cell functions using cell extracts. Nat. Biotechnol. 20,460 -466.[CrossRef][Medline]
Hussein, T. S., Thompson, J. G. and Gilchrist, R. B. (2006). Oocyte-secreted factors enhance oocyte developmental competence. Dev. Biol. 296,514 -521.[CrossRef][Medline]
Kang, Y. K., Park, J. S., Koo, D. B., Choi, Y. H., Kim, S. U., Lee, K. K. and Han, Y. M. (2002). Limited demethylation leaves mosaic-type methylation states in cloned bovine pre-implantation embryos. EMBO J. 21,1092 -1100.[CrossRef][Medline]
Kim, J. M., Liu, H., Tazaki, M., Nagata, M. and Aoki, F.
(2003). Changes in histone acetylation during mouse oocyte
meiosis. J. Cell Biol.
162, 37-46.
Kishigami, S. and Wakayama, T. (2007). Efficient strontium-induced activation of mouse oocytes in standard culture media by chelating calcium. J. Reprod. Dev. 53,1207 -1215.[CrossRef][Medline]
Kishigami, S., Hikichi, T., Van Thuan, N., Ohta, H., Wakayama, S., Bui, H. T., Mizutani, E. and Wakayama, T. (2006). Normal specification of the extraembryonic lineage after somatic nuclear transfer. FEBS Lett. 580,1801 -1806.[CrossRef][Medline]
Lee, J., Okada, K., Ogushi, S., Miyano, T., Miyake, M. and Yamashita, M. (2006). Loss of Rec8 from chromosome arm and centromere region is required for homologous chromosome separation and sister chromatid separation, respectively, in mammalian meiosis. Cell Cycle 5,1448 -1455.[Medline]
Livak, K. J. and Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25,402 -408.[CrossRef][Medline]
McGrath, J. and Solter, D. (1984). Inability of
mouse blastomere nuclei transferred to enucleated zygotes to support
development in vitro. Science
226,1317
-1319.
Miyamoto, K., Furusawa, T., Ohnuki, M., Goel, S., Tokunaga, T., Minami, N., Yamada, M., Ohsumi, K. and Imai, H. (2007). Reprogramming events of mammalian somatic cells induced by Xenopus laevis egg extracts. Mol. Reprod. Dev. 74,1268 -1277.[CrossRef][Medline]
Nichols, J., Zevnik, B., Anastassiadis, K., Niwa, H., Klewe-Nebenius, D., Chambers, I., Schöler, H. and Smith, A. (1998). Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell 95,379 -391.[CrossRef][Medline]
Obata, Y., Kono, T. and Hatada, I. (2002). Gene silencing: maturation of mouse fetal germ cells in vitro. Nature 418,497 .[CrossRef][Medline]
Ogushi, S., Palmieri, C., Fulka, H., Saitou, M., Miyano, T. and
Fulka, J., Jr (2008). The maternal nucleolus is essential for
early embryonic development in mammals. Science
319,613
-616.
Pangas, S. A. and Rajkovic, A. (2006).
Transcriptional regulation of early oogenesis: in search of masters.
Hum. Reprod. Update 12,65
-76.
Polanski, Z., Hoffmann, S. and Tsurumi, C. (2005). Oocyte nucleus controls progression through meiotic maturation. Dev. Biol. 281,184 -195.[CrossRef][Medline]
Santos, F., Zakhartchenko, V., Stojkovic, M., Peters, A., Jenuwein, T., Wolf, E., Reik, W. and Dean, W. (2003). Epigenetic marking correlates with developmental potential in cloned bovine preimplantation embryos. Curr. Biol. 13,1116 -1121.[CrossRef][Medline]
Shi, W., Hoeflich, A., Flaswinkel, H., Stojkovic, M., Wolf, E.
and Zakhartchenko, V. (2003). Induction of a senescent-like
phenotype does not confer the ability of bovine immortal cells to support the
development of nuclear transfer embryos. Biol. Reprod.
69,301
-309.
Simonsson, S. and Gurdon, J. (2004). DNA demethylation is necessary for the epigenetic reprogramming of somatic cell nuclei. Nat. Cell Biol. 6, 984-990.[CrossRef][Medline]
Strumpf, D., Mao, C. A., Yamanaka, Y., Ralston, A.,
Chawengsaksophak, K., Beck, F. and Rossant, J. (2005). Cdx2
is required for correct cell fate specification and differentiation of
trophectoderm in the mouse blastocyst. Development
132,2093
-2102.
Tada, M., Takahama, Y., Abe, K., Nakatsuji, N. and Tada, T. (2001). Nuclear reprogramming of somatic cells by in vitro hybridization with ES cells. Curr. Biol. 11,1553 -1558.[CrossRef][Medline]
Taranger, C. K., Noer, A., Sørensen, A. L.,
Håkelien, A. M., Boquest, A. C. and Collas, P. (2005).
Induction of dedifferentiation, genomewide transcriptional programming, and
epigenetic reprogramming by extracts of carcinoma and embryonic stem cells.
Mol. Biol. Cell 16,5719
-5735.
Van Thuan, N., Wakayama, S., Kishigami, S., Ohta, H., Hikichi,
T., Mizutani, E., Bui, H. T. and Wakayama, T. (2006a).
Injection of somatic cell cytoplasm into oocytes before intracytoplasmic sperm
injection impairs full-term development and increases placental weight in
mice. Biol. Reprod. 74,865
-873.
Van Thuan, N., Wakayama, S., Kishigami, S. and Wakayama, T.
(2006b). Donor centrosome regulation of initial spindle formation
in mouse somatic cell nuclear transfer: roles of gamma-tubulin and nuclear
mitotic apparatus protein 1. Biol. Reprod.
74,777
-787.
Wakayama, S., Cibelli, J. B. and Wakayama, T. (2003). Effect of timing of the removal of oocyte chromosomes before or after injection of somatic nucleus on development of NT embryos. Cloning Stem Cells 5,181 -189.[CrossRef][Medline]
Wakayama, T. (2007). Production of cloned mice and ES cells from adult somatic cells by nuclear transfer: how to improve cloning efficiency? J. Reprod. Dev. 53, 13-26.[CrossRef][Medline]
Wakayama, T., Perry, A. C., Zuccotti, M., Johnson, K. R. and Yanagimachi, R. (1998). Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature 394,369 -374.[CrossRef][Medline]
Wakayama, T., Tateno, H., Mombaerts, P. and Yanagimachi, R. (2000). Nuclear transfer into mouse zygotes. Nat. Genet. 24,108 -109.[CrossRef][Medline]
Wilmut, I., Schnieke, A. E., McWhir, J., Kind, A. J. and Campbell, K. H. (1997). Viable offspring derived from fetal and adult mammalian cells. Nature 385,810 -813.[CrossRef][Medline]
Wilmut, I., Beaujean, N., de Sousa, P. A., Dinnyes, A., King, T. J., Paterson, L. A., Wells, D. N. and Young, L. E. (2002). Somatic cell nuclear transfer. Nature 419,583 -586.[CrossRef][Medline]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
