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First published online 3 July 2008
doi: 10.1242/dev.016329
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,
1 Department of Genetic Medicine, Weill Medical College of Cornell University,
1300 York Avenue, New York, NY, USA.
2 Weill Graduate School of Medical Sciences of Cornell University, Weill Medical
College of Cornell University, 1300 York Avenue, New York, NY, USA.
3 Department of Cell and Developmental Biology, Weill Medical College of Cornell
University, 1300 York Avenue, New York, NY 10021, USA.
4 NV Organon, Oss, The Netherlands.
Author for correspondence (e-mail:
sac269{at}cornell.edu)
Accepted 26 May 2008
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Oocyte, Cytoplasmic lattice, Peptidyl arginine deiminase 6, Maternal effect gene, Ribosomal storage, Translational regulation, Embryonic genome activation, Ribosomal protein S6
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In Xenopus and Drosophila, while EGA
occurs relatively quickly in real time (<10 hours) it does not initiate
until after at least the 12th cell division. In mammals, however, while taking
more than 2-4 days to occur, EGA actually initiates after just one cell
division in the mouse and after two or three cell divisions in humans, cattle
and sheep (Schultz, 2002). Given these observations, it has remained unclear
as to whether mammalian oocytes need to maintain a storehouse of maternal
ribosomes. Nevertheless, biochemical data in the mouse indicate that 75-80% of
ribosomes are not incorporated into polysomes at ovulation and do not engage
in protein synthesis in vitro (Bachvarova
and De Leon, 1977).
Several lines of evidence suggest that these inactive ribosomes are
actually embedded in the oocyte cytoplasmic lattices (CPLs), a fibrillar
matrix composed of a proteinaceous component and RNA. Previous investigators
have observed that a precursor-product relationship exists between ribosomes
and the CPLs during oocyte growth, with the number of ribosomes decreasing as
the density of CPLs increases (Garcia et
al., 1979; Sternlicht and
Schultz, 1981; Wassarman and
Josefowicz, 1978). Furthermore, roughly two-thirds of the
ribosomes predicted to exist, based on total rRNA levels, cannot be visualized
at the ultrastructural level in fully grown oocytes
(Garcia et al., 1979;
Zamboni, 1970). The
observation that the CPL fibrils contain repeating units of electron-dense
ribosome-sized particles suggested that these `missing' ribosomes are actually
contained within the CPLs (Sternlicht and
Schultz, 1981). Finally, unlike somatic cell ribosomes, which
require centrifugal forces of at least 100,000 g (>1 hour)
for pelleting, roughly 70% of oocyte rRNA partitions in the cell pellet
following centrifugation at 9000 g (5 minutes), suggesting
that most egg ribosomes are associated with a large supramolecular complex
(Bachvarova et al., 1981). An
additional series of experiments has suggested that the lattices also contain
intermediate filaments such as keratin, leading to the hypothesis that they
represent a unique oocyte cytoskeletal element
(Capco et al., 1993;
McGaughey and Capco, 1989).
Given that many components of the protein synthetic machinery form
associations with intermediate filaments, this interpretation does not
diminish the possibility that the CPLs also contain ribosomes
(Hesketh and Pryme, 1991;
Hovland et al., 1996).
Most recently, our work has implicated peptidyl arginine deiminase 6
(PADI6), a highly abundant oocyte- and early embryo-restricted protein, in
lattice regulation. In an initial study characterizing this novel oocyte
factor, we showed by immuno-electron microscopy (immuno-EM) that PADI6
localizes primarily to the CPLs in mature oocytes
(Wright et al., 2003). Then,
in order to investigate PADI6 function, we inactivated the Padi6 gene
in mice and found that Padi6-/- females are infertile,
with the developmental arrest occurring at the two-cell stage. This
established Padi6 as a novel mammalian maternal effect gene.
Additionally, we found that the CPL structure cannot be visualized in
Padi6-/- metaphase II-arrested oocytes, suggesting that
PADI6 is required for CPL formation and/or maintenance and that the lattices
are required for development beyond the two-cell stage
(Esposito et al., 2007). This
also established the Padi6-/- mouse as an ideal model
system in which to test a number of the above hypotheses concerning lattice
function.
Here, we present findings that further strengthen the model that the CPLs are involved in ribosomal storage in oocytes and that PADI6 is critical for lattice formation. We also show that, in Padi6-/- two-cell embryos, ribosomal component levels are reduced and de novo protein synthesis is dysregulated. Finally, we establish that the mechanism of the two-cell arrest in Padi6-/- embryos is probably due to a failure to completely activate the embryonic genome. Taken together, these findings underscore the potential importance of lattices in mammalian early development.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). All germinal vesicle (GV) stage oocytes and two-cell
embryos were collected from 3- to 5-week-old Padi6+/+ and
Padi6-/- female mice. GV stage oocytes were isolated from
ovarian follicles 
46 hours after PMSG (5 IU) stimulation and two-cell
embryos were isolated from the oviducts of superovulated and mated female mice
44 hours after hCG (5 IU) treatment.
Electron microscopy
For EM analysis of primordial follicle and growing oocytes, ovaries were
collected from 14-day-old and 7-week-old females, respectively. They were
fixed for 2 hours in 2.5% glutaraldehyde, 4% paraformaldehyde and 0.1% tannic
acid in 0.1 M sodium cacodylate buffer (pH 7.3) (TA). GV oocytes and two-cell
embryos were collected as described above, washed in PBS, pipetted onto
polylysine-coated coverslips and fixed in TA for 2 hours. The cells were then
incubated for 1 hour with 1% osmium tetroxide and stained with uranyl acetate
en bloc. The eggs were then dehydrated through a series of graded ethanols and
embedded in Spurr's resin (Electron Microscopy Sciences, Hatfield, PA).
Semi-thin (0.5 µm), then ultra-thin (55-60 nm, silver-gold) sections were
cut using a Diatome diamond knife on a Leica Ultracut S Ultramicrotome (Leica,
Vienna, Austria), contrasted with lead citrate and viewed on a JEM 100 CX-II
electron microscope (JEOL, USA, Peabody, MA) operated at 80 kV. Images were
recorded on Kodak 4489 Electron Image film then digitized on an Epson
Expression 1600 Pro scanner at 1200 dpi for publication. For electron
microscopy analysis of the 9000 g pellet, 20
Padi6+/+ and Padi6-/- GV stage oocytes
were ruptured in 200 µl of rupture buffer and centrifuged as described
below. The cell pellets were fixed in 2% glutaraldehyde in 0.05 M sorenson
phosphate buffer and encapsulated in 1% agarose. Samples were then rinsed in
buffer and post-fixed in 1% OsO4. Next, the agarose pellets were
released from centrifuge tubes, excess agarose was trimmed away, and the
samples were processed as above.
Western blot and qRT-PCR analysis
For western blotting, Padi6+/+ and
Padi6-/- GV stage oocytes (30) and two-cell embryos (30)
were isolated, boiled for 5 minutes in Laemmli buffer, and directly loaded
onto a 10% SDS-PAGE gel. Proteins were separated at 200 V for 50 minutes and
then transferred to a nitrocellulose membrane by applying current of 90 V for
90 minutes. All blots were blocked with 5% nonfat dry milk in TBS containing
0.5% Tween-20 (TBS-T), washed and incubated with a 1:1000 dilution of
anti-ribosomal S6 sera (rabbit mAb, Cell Signaling, Danvers, MA) in 5% BSA or
anti-tubulin sera (1:1000 dilution, Millipore, Temecula, CA) in blocking
buffer overnight at 4°C. The blots were then washed three times for 10
minutes in TBS-T and incubated with 1:10,000 dilution of peroxidase-conjugated
goat anti-rabbit IgG or donkey anti-mouse IgG secondary antibody (Jackson
ImmunoResearch, West Grove, PA) for 1 hour. Following incubation in secondary
antibody, the membranes were washed three times for 10 minutes in TBS-T, and
Immobilon Western HRP Chemiluminescent Substrate (Millipore, Temecula, CA) was
applied for 5 minutes and developed. The experiments were repeated three
times.
For real-time RT-PCR quantitation of mRNAs, total RNA was isolated from 35
GV stage oocytes using Trizol and chloroform. The RNA contained within the
aqueous fraction was then purified using RNeasy Mini Kit (Qiagen, Valencia,
CA), reverse transcribed, and subjected to real-time PCR using
sequence-specific 18S rRNA primers for TaqMan Gene Expression Assays (Applied
Biosystems, Foster City, CA). PCR was performed using the TaqMan PCR Master
Mix and the ABI 7700 thermal cycler (Applied Biosystems) using the following
parameters: 50°C for 2 minutes and 95°C for 10 minutes, followed by 40
cycles at 95°C for 15 seconds and 60°C for 1 minute. 18S rRNA
expression levels were normalized to Gapdh mRNA and quantitated using the
2-
CT method.
Oocyte fractionation and immuno slot-blot analysis
For sedimentation analysis, Padi6+/+ and
Padi6-/- GV oocytes were isolated from follicles and
immediately ruptured in 200 µl of a buffer containing 0.05 M KCl, 0.2 M
sucrose, 50 mM KCl, 0.5% Triton X-100, 10 mM HEPES (pH 7.3) and complete
protease inhibitor cocktail (Roche, Branchburg, NJ, USA) using a 60 µm
(inner diameter) pulled Pasteur pipette. The lysate was then centrifuged at
650 g for 5 minutes at room temperature and the supernatant
was centrifuged again at 9000 g for 5 minutes. The supernatant
and pellet fractions were then incubated on ice in lysis buffer (50 mM Tris
HCl, 150 mM NaCl, 1% NP40, and protease inhibitors) for 2 hours, heated to
100°C for 5 minutes in Laemmli buffer and directly loaded onto a PVDF
membranes (Immobilon, Millipore, Billerica, MA) using a slot-blot apparatus
(Hoefer, San Francisco, CA). The blots were then probed with anti-S6 and
anti-PADI6 antibodies as described above.
Scanning confocal and wide-field microscopy, and immunofluorescence analysis
Following collection as described above, oocytes and embryos were either
immediately fixed in 4% paraformaldehyde in Dulbecco's PBS (DPBS) (Invitrogen)
for 30 minutes at room temperature or incubated first for 20 minutes in
extraction buffer (0.1 M KCl, 20 mM MgCl2, 3 mM EGTA, 20 mM HEPES
(pH 6.8), 1% Triton X-100, Complete Protease Inhibitor Cocktail (Roche,
Mannheim, Germany) then rinsed quickly three times in DPBS. The
oocytes/embryos were then washed five times in IF buffer (DPBS, 1% BSA, 0.5%
NGS), permeabilized with 0.5% Triton X-100 in PBS for 30 minutes, washed again
(five times in IF), and incubated with guinea pig anti-PADI6 (1:500)
(Wright et al., 2003), rabbit
anti-S6 (1:200), rabbit anti-RNA Pol II (1:100, N-20, Santa Cruz
Biotechnology), mouse anti-RNA Pol II CTD phospho-serine 2 (1:400, H5, Covance
Research Products, Berkeley, CA), rabbit anti-spindlin (1:200, a generous gift
from Barbara Knowles, The Jackson Laboratory, Bar Harbor Maine), rabbit
anti-DNMT1 (1:400, PATH52 antiserum a generous gift from Timothy Bestor) or
anti-H4K5 acetyl (1:200, Millipore, Billerica, MA) antibody in IF buffer
overnight at 4°C. Oocytes/embryos were washed again (five times in IF) and
incubated for 2 hours at room temperature with a 1:450 dilution of the
appropriate secondary antibody: goat anti-guinea pig Alexa Fluor 546, goat
anti-rabbit Alexa Fluor 488 or goat anti-mouse Alexa Fluor 633 (Molecular
Probes, Eugene, OR). Oocytes and embryos were mounted on slides in Slowfade
Gold antifade reagent (Molecular Probes, Eugene, OR), and imaged using an LSM
510 laser scanning confocal microscope (Zeiss). Extensive optimization was
performed while imaging to ensure that we were as near to the theoretical
resolution limit (200 nm) as possible. Data were analyzed with MetaMorph
7 (Molecular Devices, Downingtown, PA) and colocalization analysis was
performed using Volocity 4 (Improvision, Lexington, MA). Using MetaMorph7 for
quantification of nuclear markers in Fig.
5A, a region of interest was defined using the DAPI signal as a
marker for nuclear boundaries. Then the average intensity of the region for
the different channels was logged to an Excel file where the data were
averaged and statistics could be calculated. Once the average was calculated
for each group [H4K5acetyl: average intensity 2286.3 (n=32)
Padi6+/+ versus 2058.5 (n=24)
Padi6-/- t-test two-tailed P=0.023; phospho-pol
II: average intensity 935.6 (n=16) Padi6+/+
versus 626.8 (n=23) Padi6-/- t-test two-tailed
P=0.006; pol II: average intensity 1572 (n=16)
Padi6+/+ versus 1272 (n=22)
Padi6-/- t-test two-tailed P=0.003], an image
most closely matching the average signal intensity was chosen as a
`representative' nucleus. For DNMT1 localization, oocytes were imaged at
1000x using a Zeiss Axiovert-200 fluorescence microscope.
Total protein synthesis and resolution of radiolabeled proteins
GV oocytes and two-cell embryos were radiolabeled for 2 hours in CZB medium
containing 1 mCi/ml of [35S]methionine (>1000 Ci/mMol; Perkin
Elmer, Shelton, CT, USA). Following radiolabeling, the embryos were washed in
MEM/PVA, then in PBS/PVA and frozen in liquid nitrogen until use. Total
protein was precipitated for 1 hour using 10% trichloroacetic acid (TCA)
dissolved in 1 M NaOH and assayed by scintillation counting. To visualize de
novo protein synthesis patterns, 20 GV oocytes and 20 two-cell embryos were
resolved using 10% SDS-PAGE as previously described
(Conover et al., 1991). After
electrophoresis, the gel was dried and exposed overnight to a phosphor imager
screen. The radiolabeled proteins were then visualized using a Typhoon Triad
PhosphorImager.
Transcription requiring complex analysis
Two-cell embryos were collected at 43-46 hours post hCG injection and
radiolabeled for 2 hours in CZB medium containing 1 mCi/ml of
[35S]methionine (>1000 Ci/mMol; Perkin Elmer, Shelton, CT, USA).
Following radiolabeling, the embryos were washed in MEM/PVA, treated
for 10 minutes (room temperature) in a solution containing 50 mM Tris-HCl (pH
7.4), 2% Triton-X100 and 0.3 M KCl to enrich for the TRC proteins. Labeled
proteins were then resolved by SDS-PAGE (10% gel) as described previously
(Conover et al., 1991). The
radiolabeled proteins were visualized using a Typhoon Triad PhosphorImager.
TRC protein levels were quantified from tif files of the fluorograph by
calculating the intensity of protein signal using the Photoshop Histogram
Analysis program.
BrUTP incorporation assay
BrUTP incorporation assays were performed essentially as previously
described (Aoki et al., 1997).
Briefly, two-cell embryos were permeabilized for 2 minutes with 0.05% Triton
X-100 in PB: 100 mM potassium acetate, 30 mM KCl, 1 mM MgCl2, 10 mM
Na2HPO4, 1 mM ATP, 1 mM DTT, supplemented with 1x
complete protease inhibitor cocktail (Roche, Branchburg, NJ, USA) and 50
units/ml of RNasin. Treated embryos were then washed in PB and incubated for
10 minutes at 33°C in 100 mM potassium acetate, 30 mM KCl, 2 mM
MgCl2, 10 mM Na2HPO4, 2 mM ATP, 0.4 mM each
of GTP, CTP and BrUTP. Next, embryos were washed in PB and the nuclear
membrane was permeabilized in PB containing 0.2% Triton X-100 for 3 minutes.
The embryos where washed again in PB and fixed with 4% paraformaldehyde for 30
minutes. BrUTP incorporation was detected by immunostaining using a 1:200
dilution of antiBrdU monoclonal antibody (Chemicon, Temecula, CA, USA). The
embryos were then incubated with a 1:200 dilution of donkey anti-mouse
FITC-labeled secondary antibody (Jackson ImmunoResearch) and processed as
indicated in the immunofluorescence section. Following image capture, the
signal was quantified from .tif files of the embryos using the Photoshop
Histogram Analysis program by calculating the pixel value/unit area of five
different random regions of the nucleus and five different region of the
cytoplasm and by subtracting the average cytoplasmic value from the average
nucleoplasm value. The transcription levels of the
Padi6-/- nuclei were expressed as a percentage of the
Padi6+/+ transcription levels.
Statistical analysis
All experiments used for quantification were repeated at least three times.
Statistical significance was assessed using the Microsoft Excel 2003 program
using a two-tailed t-test.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Similar to previous reports
(Zamboni, 1970), we next found
that the CPLs first become evident in Padi6+/+ oocytes at
approximately the 40 µm stage of growth and appear to be derived from
curvilinear `intermediate structures' (IS), which have properties of both
aggregated ribosomes (Rb) and mature lattices
(Fig. 1C). However, no CPLs or
IS were observed in 40 µm Padi6-/- oocytes
(Fig. 1D). CPLs were also
evident in fully grown germinal vesicle (GV) stage
Padi6+/+ oocytes (Fig.
1E), whereas CPLs were not observed in similarly staged
Padi6-/- oocytes (Fig.
1F). Non-lattice-associated ribosome levels appeared similar
between stage-matched Padi6+/+ and
Padi6-/- oocytes. We found that CPLs were also not
observed in Padi6-/- two-cell embryos
(Fig. 1H) whereas lattices in
Padi6+/+ two-cell embryos were visible (arrows,
Fig. 1G). As lattice structures
are never detected at any stage of oocyte growth in
Padi6-/- oocytes, we conclude that PADI6 is indeed
required for lattice formation.
Overall levels of ribosomal components are not altered in Padi6-/- oocytes
To further evaluate the relationship between PADI6 and ribosomes, we next
investigated whether total ribosome levels were altered in
Padi6-/- oocytes. Analysis of 40S ribosomal subunit levels
by quantitative real time(q) RT-PCR using 18S rRNA probes and by immunoblot
analysis using an antibody reactive with the small ribosomal subunit protein,
S6, found that loss of PADI6 does not affect levels of these ribosomal
components (Fig. 2A).
Therefore, lack of lattice formation does not appear to have consequences on
overall ribosomal levels in the oocyte.
Ribosomal components display increased solubility in Padi6-/- oocytes
Given previous observations that 70% of oocyte rRNA can be pelleted at low
centrifugal force (9000 g), and therefore is most likely
associated with a large supramolecular complex, we next wanted to test whether
the majority of ribosomal S6 protein also pellets at 9000 g.
Furthermore, we reasoned that if the lattices are indeed the supramolecular
complex associated with stored ribosomal components, S6 should pellet less
readily in Padi6-/- oocytes. Accordingly, we ruptured
Padi6+/+ and Padi6-/- oocytes in a
hypotonic 0.05 M KCl buffer containing 0.2 M sucrose and 0.5% Triton X-100,
and serially centrifuged the oocyte lysates for 5 minutes at 650 and 9000
g. Ribosomal protein levels were then evaluated by
immuno-slot-blot analysis using the S6 antibody. Results show that the
majority of the S6 protein partitioned in either the 650 or 9000
g pellet in Padi6+/+ oocytes
(Fig. 2B). This finding further
supports the hypothesis that, in mammalian oocytes, ribosomal components are
associated with a supramolecular complex. In Padi6-/-
oocytes, however, most of the S6 protein partitioned in the supernatant
fraction following rupture and centrifugation
(Fig. 2B). We next performed
TEM analysis of the Padi6+/+ and
Padi6-/- 9000 g oocyte lysate pellets to
test the hypothesis that the CPLs represent this complex. Results show that
the major distinction between these two samples was that, although an
abundance of lattice-like structures were found in the
Padi6+/+ oocyte pellet (arrow,
Fig. 2C), these structures were
not observed in the Padi6-/- oocyte pellet
(Fig. 2D). Interestingly,
ribosomes appear to be directly associated with some of the putative CPL
structures (arrowheads). In further support of the hypothesis that ribosomes
are contained within CPLs, we also found by immuno-slot-blot analysis that
PADI6, which localizes to CPLs, mainly partitions in the 9000
g pellet of Padi6+/+ oocyte lysates (data
not shown). Taken together, the above findings support the hypothesis that the
CPLs contain ribosomal components and that PADI6 is required for their
incorporation.
|
; McGaughey and
Capco, 1989), and we found that Triton extraction before fixation
allowed us to test more reliably for colocalization. Similar techniques are
used for visualization of proteins associated with the cytoskeleton
(Spector, 1998). Results show
that, in Triton extracted oocytes, PADI6 and S6 appear to colocalize in
regions throughout the cytoplasm and at granular foci in the oocyte cortex
(Fig. 3A). We further
controlled for random colocalization owing to high abundance of both factors
by observing the extent of colocalization before and after a 20 pixel shift of
one of the channels with respect to the other. Random colocalization should
not be dramatically affected by a pixel shift. We observed a reproducible
reduction in colocalization by 30% after the pixel shift, therefore, at least
30% of our observed colocalization is not due to random overlap. Given that
the theoretical resolution limit of our confocal analysis (200 nm) is
larger than the repeating units of the lattices (21nm), we next attempted
to localize the S6 ribosomal protein to the lattices by immuno-EM.
Unfortunately, however, we were not able to obtain specific labeling of
oocytes using either the S6 antibody or other commercially available ribosomal
protein antibodies. Thus, we proceeded with our confocal analysis, as it was
the highest resolution method available.
Confocal analysis further revealed that S6 is retained in the insoluble
fraction in Triton extracted Padi6-/- oocytes; however,
the staining is more punctate than in extracted Padi6+/+
oocytes. Most striking, however, is the complete absence of S6 from the cortex
of Padi6-/- oocytes (arrow,
Fig. 3A). It has been observed
that a number of other proteins, including actin
(Sun and Schatten, 2006) and
DNMT1 (Ratnam et al., 2002),
also abundantly localize to the oocyte cortex. To test whether the
localization of these factors is also affected by loss of PADI6, we first
probed intact oocytes with an antibody specific for DNMT1. We found by
wide-field immunofluorescence microscopy that DNMT1 localization does not
appear to be affected in Padi6-/- oocytes
(Fig. 3C). Because actin is
Triton insoluble, we then probed Triton-extracted oocytes with phalloidin and
found that the actin network in the cortex is intact in
Padi6-/- oocytes (Fig.
3B). Therefore, we predict that PADI6 and/or the CPLs are
responsible for the specific targeting of ribosomal components to the oocyte
cortex, and that, in their absence, proper targeting does not occur. Taken
together, these results further strengthen the interrelationship between
PADI6, ribosomal components and the CPLs.
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), it is also possible that these ribosomes play a role in
protein synthesis in oocytes. To test this hypothesis, we metabolically
labeled Padi6+/+ and Padi6-/- GV stage
oocytes for 2 hours using [35S]methionine and evaluated de novo
protein synthesis by scintillation counting and fluorography of resolved
proteins. Results showed that there were no reliable differences in overall
translation levels (data not shown) and, with few exceptions, the repertoire
of nascently synthesized proteins appears similar between
Padi6+/+ and Padi6-/- oocytes
(Fig. 3D). The 72 kDa protein
(arrow) missing from Padi6-/- oocytes is probably PADI6.
This result, coupled with the observation that oocyte growth and fertilization
are not affected by loss of PADI6, supports the hypothesis that putative
CPL-associated ribosomal components do not play a crucial role in protein
translation in oocytes.
S6 protein levels are lower and localization is dramatically altered in Padi6-/- two cell embryos
Our previous finding that Padi6-/- embryos arrest at
the two-cell stage suggested that putative CPL-associated ribosomal components
are probably required for protein synthesis in the early embryo. To begin
testing this hypothesis, we next evaluated S6 levels and localization by
western blot and confocal immunofluorescence analysis in
Padi6+/+ and Padi6-/- two-cell
embryos. Results show that both PADI6 and S6 strongly colocalize at the
non-apposed cortical regions of each blastomere in
Padi6+/+ embryos (Fig.
4A). In Padi6-/- two-cell embryos, we found
that total levels of S6 were reduced (Fig.
4A,D) and that S6 is not enriched at embryonic cortex. These
observations suggest that, in contrast to the oocyte, PADI6 appears to be
crucial for helping maintain proper ribosomal levels in the two-cell embryo.
Furthermore, similar to our finding in oocytes, these observations suggest
that PADI6 also appears to be required for localization of S6 to the embryonic
cortex (arrows, Fig. 4C). The
microfilament network is intact throughout the entire cortex region of
blastomeres in both Padi6+/+ and
Padi6-/- two-cell embryos
(Fig. 4B), indicating that
PADI6 only appears to be required for targeting a specific subset of molecules
to the embryonic cortex.
De novo protein synthesis is altered in Padi6-/- two cell embryos
To test the hypothesis that reduced levels and disrupted localization of S6
in Padi6-/- two-cell embryos affects protein translation,
we next metabolically labeled the embryos with [35S]methionine for
2 hours and then evaluated de novo protein synthesis by scintillation counting
and by fluorography of resolved proteins. Results show that total protein
synthesis levels were reduced by 50% in Padi6-/-
two-cell embryos compared with Padi6+/+ embryos (data not
shown). In addition to reduced global levels of protein synthesis,
fluorographic analysis of newly synthesized proteins found that expression
levels of specific proteins in Padi6-/- two-cell embryos
are altered when compared with Padi6+/+ embryos
(Fig. 4E). In particular,
synthesis of one protein at 30 kDa is dramatically upregulated in the
Padi6-/- two-cell embryo. Based on previous reports, we
hypothesized that this protein was probably spindlin, the product of a stored
maternal transcript thought to be involved in regulating the cell cycle during
meiosis and the first mitotic cell division
(Oh et al., 1997). We next
tested the hypothesis that spindlin protein synthesis is dysregulated in
Padi6-/- two-cell embryos by confocal analysis using
anti-spindlin antisera and found that spindlin levels were, in fact, increased
in Padi6-/- two-cell embryos
(Fig. 4F). It is currently
unclear why, in addition to the observed increase in cytoplasmic spindlin
levels in Padi6-/- two-cell embryos, levels of spindlin
also appear to be increased in the perivitelline space of these embryos;
however, this may be due to leakage of the protein during fixation.
Taken together, these observations support the hypothesis that improper CPL formation in Padi6-/- oocytes leads to an overall reduction in protein synthesis in two-cell embryos. Furthermore, the altered translation pattern in Padi6-/- two-cell embryos suggests that the CPL complex may also play a role in organizing and regulating the translational machinery such that specific maternal mRNAs are translated at different efficiencies in Padi6-/- embryos compared with Padi6+/+ embryos. The observation that spindlin levels are increased in Padi6-/- embryos also suggests that the observed overall reduction in total protein synthesis is not simply due to cell death.
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).
Therefore, we next tested the hypothesis that the observed translational
defects in Padi6-/- two-cell embryos lead to defective
EGA. In mammals, nuclear translocation of maternally inherited RNA Polymerase
II (RNA Pol II) is thought to be a major determinant of EGA
(Bellier et al., 1997).
Therefore, we first investigated whether RNA Pol II levels and/or localization
was specifically affected by loss of PADI6. We probed two-cell embryos with an
antibody that recognizes the N terminus of RNA Pol II and found that levels of
RNA Pol II were reduced by 20% in the nucleus of
Padi6-/- two-cell embryos when compared with
Padi6+/+ embryos (Fig.
5A). Furthermore, we also found that levels of phosphorylated (Ser
2) RNA Pol II (a well characterized marker for active transcription)
(Palancade and Bensaude, 2003)
were reduced by 30% in the nucleus of Padi6-/-
embryos (Fig. 5A). These
results suggest that both RNA Pol II nuclear translocation and RNA Pol
II-mediated transcription are reduced in Padi6-/- embryos.
In addition, our metabolic labeling data suggests that the basis for the
nuclear translocation deficiency may lie in the improper translation of
factors required for the transport of RNA Pol II from the cytoplasm to the
nucleus. This hypothesis is supported by the observation that RNA Pol II
appears to aggregate outside of the nuclear envelope in
Padi6-/- embryos (Fig.
5A). Given that histone H4 acetylation is well correlated with
active gene transcription (Roth et al.,
2001), we next probed Padi6+/+ and
Padi6-/- two-cell embryos with an anti-histone H4 acetyl
antibody (histone H4K5) as a further test of the effects of loss of PADI6 on
embryonic transcription. Results show a small, but statistically significant,
10% reduction in histone H4 acetylation in Padi6-/-
two-cell embryo nuclei when compared with Padi6+/+ embryos
(Fig. 5A). We next measured
nuclear incorporation of BrUTP in Padi6+/+ and
Padi6-/- two-cell embryos using an anti-BrUTP antibody and
fluorescence microscopy (Aoki et al.,
1997). Fluorescence analysis showed that transcription levels were
reduced by 73.7% in Padi6-/- embryos compared with
Padi6+/+ embryos (Fig.
5B). Finally, we investigated the de novo synthesis of the
transcription requiring complex (TRC), a group of Triton X-100 insoluble
proteins that are the first major products of embryonic transcription and thus
function as a marker for EGA (Schultz et
al., 1999). Results show that TRC synthesis is reduced by 53%
in Padi6-/- two-cell embryos when compared with
Padi6+/+ embryos (Fig.
5C), thus further supporting the hypothesis that
Padi6-/- two-cell embryos fail to undergo complete
embryonic genome activation. Interestingly, others have noted that inhibiting
embryonic transcription (as evaluated by TRC synthesis) also inhibits the
development-associated decrease in spindlin synthesis
(Worrad et al., 1995); thus,
our observation of the inverse relationship between embryonic transcription
and spindlin synthesis is in line with previous findings.
|
|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Our findings support the hypothesis that improper CPL formation
Padi6-/- oocytes affects ribosomal storage leading to
overall lower levels of de novo protein synthesis in the two-cell embryo and
to dysregulation of the translational program. Furthermore, we predict that
this change in protein synthesis leads to incomplete activation of the
embryonic genome and, subsequently, arrest at the two-cell stage. Our results
also demonstrate that the mechanism of the Padi6-/-
two-cell arrest actually initiates during oocyte growth. Previously, we
reported an absence of lattices in ovulated mature
Padi6-/- oocytes
(Esposito et al., 2007).
However, considering the number of dramatic ultrastructural rearrangements
that occur between oocyte growth and maturation, it was possible that PADI6
was necessary for lattice maintenance and not formation. Here, we undertook a
more comprehensive ultrastructural study of lattice formation from the
primordial oocyte stage until the two-cell embryo. In agreement with previous
studies, we found in Padi6+/+ oocytes that the lattices
first become apparent in the growing oocyte. Additionally, we were able to
detect all of the putative steps of lattice formation from aggregated
polysomes and curvilinear intermediate structures to elongated linear
lattices. In striking contrast, however, we never observed any stages of
lattice formation in any Padi6-/- oocytes. Thus, we
conclude that PADI6 is necessary for the earliest stages of lattice
formation.
|
Previous reports found that, unlike in somatic cells, the majority of rRNA
in murine oocyte is pelleted at low centrifugal forces (9000
g). This finding lead to the conclusion that oocyte ribosomes
are associated with a large supramolecular complex, most probably the lattices
(Bachvarova et al., 1981;
Brower and Schultz, 1982). In
our current study, we strengthened the validity these reports of by showing
that the ribosomal S6 protein readily pellets at 9000 g in
Padi6+/+ oocytes. If the CPLs were indeed the
supramolecular structure with which the ribosomal components are associated,
then one would expect the S6 protein to not pellet as readily in the
Padi6-/- oocytes. This is indeed what we observed.
Furthermore, our EM results showed that the major difference between the 9000
g pellets of Padi6+/+ and
Padi6-/- oocytes is the presence or absence of the
lattices, respectively. Thus, we concluded that ribosomal components are
probably associated with the lattices, and PADI6 is required for their
incorporation. Our immunofluorescence analysis further strengthens this
hypothesis. Probing Triton-extracted oocytes with antibodies specific for S6
and PADI6, we were able to demonstrate considerable colocalization.
Our immunofluorescence data also shows that, at the two-cell stage, PADI6 and S6 abundantly localize to the microfilament-rich embryo cortex. Our original immuno-EM data localizing PADI6 to the lattices was carried out in oocytes, so it is possible that, at the two-cell stage, PADI6 is targeted from the lattices to the microfilament network in the embryo cortex. Interestingly, in the absence of PADI6, S6 localization to the cortex is completely disrupted. This observation suggests that PADI6 may also play an important role in the proper localization of translational machinery in the early embryo.
Our [35S]methionine labeling experiments show that protein
translation is reduced in Padi6-/- embryos. Furthermore,
we also found that the relative translation efficiency of specific embryonic
transcripts is altered. The most dramatic example is spindlin, which is
synthesized at relatively much higher levels in Padi6-/-
embryos. Spindlin has previously been characterized in mammalian oocytes as a
stored maternal transcript (Oh et al.,
2000). This observation supports a model in which the lattices
play a role not only in post-fertilization ribosome stability but also in
translational control of certain stored maternal transcripts. Additionally,
our observation of an overall reduction in protein synthesis in
Padi6-/- embryos implies that failing to regulate maternal
ribosomes properly does not directly lead to a global upregulation of all
stored maternal messages. This would not be surprising, given the highly
regulated nature of maternal mRNP complexes. Given that
Padi6-/- embryos arrest at the two-cell stage and that EGA
is incomplete in Padi6-/- embryos, we predict that some
the affected maternal transcripts encode key reprogramming factors. In our
study, we observed that although abundant in the cytoplasm, RNA Pol II seems
to aggregate in foci surrounding the nuclear envelope in
Padi6-/- embryos. Thus, it is possible that one of the
CPL-associated transcripts encodes a factor(s) that is/are responsible for
targeting RNA Pol II to the nucleus. In addition, given that both S6 and PADI6
localize to, and become enriched in, the cortex of two-cell embryos, and that
this enrichment is absent in Padi6-/- embryos, the cortex
may represent a special organizing site for translation of maternal
transcripts.
It is known that a significant fraction of the RNA synthesized during EGA is ribosomal RNA. As our characterizations show, a number of markers of active mRNA transcription are reduced by 10 to 30% in Padi6-/- embryos. Overall RNA synthesis, however, assayed by comparing BrUTP incorporation levels, is down by almost 75% in Padi6-/- embryos. Therefore, an alternative interpretation of the lower S6 levels in the two-cell Padi6-/- embryos is that nascent ribosomes are not being properly synthesized. The dramatically lower level of RNA synthesis, therefore, probably includes rRNAs, tRNAs, and non-coding regulatory RNAs in addition to mRNAs.
The mechanism of EGA in mammals is still not well understood. Our finding
that the Padi6-/- developmental arrest occurs at the
two-cell stage adds Padi6 to a short list of known mammalian maternal
effect genes. Interestingly, loss of most of the other known maternal effect
genes such as MATER, Zar1, NPM2, Bnc1, Hsf1 and BRG1 also results in embryonic
arrest around the two-cell stage of development
(Bultman et al., 2006;
Burns et al., 2003;
Christians et al., 2000;
Ma et al., 2006;
Tong et al., 2004;
Wu et al., 2003). EGA defects
(as determined by measuring TRC levels) have been shown to underlie the
developmental arrest phenotype of many of these maternal effect gene mutants;
however, the detailed mechanisms leading to the EGA defect in these animals
remain to be elucidated. Our finding that TRC levels are reduced by more than
50% in Padi6-/- embryos, suggests that EGA failure also
underlies the Padi6-/- developmental arrest.
In summary, in this report we provide the first mechanistic study elucidating how a mammalian maternal effect gene such as PADI6 mediates activation of the embryonic genome. We take advantage of the absence of cytoplasmic lattices in Padi6-/- oocytes and embryos to first strengthen the model that the lattices represent a storage structure for maternal ribosomes. Furthermore, we also show that the levels and localization of ribosomal components are affected in Padi6-/- two-cell embryos, as is de novo protein synthesis. Additionally, we find that the relative expression levels of specific transcripts, including a well-characterized stored maternal transcript, are also altered in these embryos. These findings lead to the prediction that improper storage of ribosomal components in Padi6-/- oocytes during oocyte growth, leads to decreased levels of ribosomes and to a dysregulated translational program in Padi6-/- two-cell embryos. Furthermore, we predict that the altered protein synthesis pattern in Padi6-/- two-cell embryos then leads to a dramatic reduction in embryonic transcriptional activity and ultimately to embryonic arrest. Alternatively, however, it is possible that the lattices do not play a direct role in early developmental events and that PADI6 alone required for EGA and development beyond the two-cell stage. Studies to uncouple the relative contributions of lattice versus PADI6 loss to the Padi6-/- two-cell arrest are ongoing.
|
ACKNOWLEDGMENTS |
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|
Footnotes |
|---|
Present address: Baker Institute for Animal Health, College of Veterinary
Medicine, Cornell University, Ithaca, NY 14850, USA
|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Aoki, F., Worrad, D. M. and Schultz, R. M. (1997). Regulation of transcriptional activity during the first and second cell cycles in the preimplantation mouse embryo. Dev. Biol. 181,296 -307.[CrossRef][Medline]
Bachvarova, R. and De Leon, V. (1977). Stored and polysomal ribosomes of mouse ova. Dev. Biol. 58,248 -254.[CrossRef][Medline]
Bachvarova, R., De Leon, V. and Spiegelman, I. (1981). Mouse egg ribosomes: evidence for storage in lattices. J. Embryol. Exp. Morphol. 62,153 -164.[Medline]
Bellier, S., Chastant, S., Adenot, P., Vincent, M., Renard, J. P. and Bensaude, O. (1997). Nuclear translocation and carboxyl-terminal domain phosphorylation of RNA polymerase II delineate the two phases of zygotic gene activation in mammalian embryos. EMBO J. 16,6250 -6262.[CrossRef][Medline]
Brower, P. T. and Schultz, R. M. (1982). Biochemical studies of mammalian oogenesis: possible existence of a ribosomal and poly(A)-containing RNA-protein supramolecular complex in mouse oocytes. J. Exp. Zool. 220,251 -260.[CrossRef][Medline]
Bultman, S. J., Gebuhr, T. C., Pan, H., Svoboda, P., Schultz, R.
M. and Magnuson, T. (2006). Maternal BRG1 regulates zygotic
genome activation in the mouse. Genes Dev.
20,1744
-1754.
Burns, K. H., Viveiros, M. M., Ren, Y., Wang, P., DeMayo, F. J.,
Frail, D. E., Eppig, J. J. and Matzuk, M. M. (2003). Roles of
NPM2 in chromatin and nucleolar organization in oocytes and embryos.
Science 300,633
-636.
Capco, D. G., Gallicano, G. I., McGaughey, R. W., Downing, K. H. and Larabell, C. A. (1993). Cytoskeletal sheets of mammalian eggs and embryos: a lattice-like network of intermediate filaments. Cell Motil. Cytoskel. 24, 85-99.[CrossRef][Medline]
Christians, E., Davis, A. A., Thomas, S. D. and Benjamin, I. J. (2000). Maternal effect of Hsf1 on reproductive success. Nature 407,693 -694.[CrossRef][Medline]
Conover, J. C., Temeles, G. L., Zimmermann, J. W., Burke, B. and Schultz, R. M. (1991). Stage-specific expression of a family of proteins that are major products of zygotic gene activation in the mouse embryo. Dev. Biol. 144,392 -404.[CrossRef][Medline]
Davidson, E. R. (1986). In Gene Activity in Early Development. Orlando, FL: Academic Press.
Esposito, G., Vitale, A. M., Leijten, F. P., Strik, A. M., Koonen-Reemst, A. M., Yurttas, P., Robben, T. J., Coonrod, S. and Gossen, J. A. (2007). Peptidylarginine deiminase (PAD) 6 is essential for oocyte cytoskeletal sheet formation and female fertility. Mol. Cell. Endocrinol. 273,25 -31.[CrossRef][Medline]
Garcia, R. B., Pereyra-Alfonso, S. and Sotelo, J. R. (1979). Protein-synthesizing machinery in the growing oocyte of the cyclic mouse. A quantitative electron microscopic study. Differentiation 14,101 -106.[CrossRef][Medline]
Hesketh, J. E. and Pryme, I. F. (1991). Interaction between mRNA, ribosomes and the cytoskeleton. Biochem. J. 277,1 -10.[Medline]
Hovland, R., Hesketh, J. E. and Pryme, I. F. (1996). The compartmentalization of protein synthesis: importance of cytoskeleton and role in mRNA targeting. Int. J. Biochem. Cell Biol. 28,1089 -1105.[CrossRef][Medline]
Ma, J., Zeng, F., Schultz, R. M. and Tseng, H.
(2006). Basonuclin: a novel mammalian maternal-effect gene.
Development 133,2053
-2062.
McGaughey, R. W. and Capco, D. G. (1989). Specialized cytoskeletal elements in mammalian eggs: structural and biochemical evidence for their composition. Cell Motil. Cytoskel. 13,104 -111.[CrossRef][Medline]
Oh, B., Hwang, S. Y., Solter, D. and Knowles, B. B. (1997). Spindlin, a major maternal transcript expressed in the mouse during the transition from oocyte to embryo. Development 124,493 -503.[Abstract]
Oh, B., Hwang, S., McLaughlin, J., Solter, D. and Knowles, B. B. (2000). Timely translation during the mouse oocyte-to-embryo transition. Development 127,3795 -3803.[Abstract]
Palancade, B. and Bensaude, O. (2003). Investigating RNA polymerase II carboxyl-terminal domain (CTD) phosphorylation. Eur. J. Biochem. 270,3859 -3870.[Medline]
Ratnam, S., Mertineit, C., Ding, F., Howell, C. Y., Clarke, H. J., Bestor, T. H., Chaillet, J. R. and Trasler, J. M. (2002). Dynamics of Dnmt1 methyltransferase expression and intracellular localization during oogenesis and preimplantation development. Dev. Biol. 245,304 -314.[CrossRef][Medline]
Roth, S. Y., Denu, J. M. and Allis, C. D. (2001). Histone acetyltransferases. Annu. Rev. Biochem. 70,81 -120.[CrossRef][Medline]
Schultz, R. M., Davis, W., Jr, Stein, P. and Svoboda, P. (1999). Reprogramming of gene expression during preimplantation development. J. Exp. Zool. 285,276 -282.[CrossRef][Medline]
Spector, D. (1998). Cells, A Laboratory Manual. Cold Spring Harbor, New York: Cold Spring Harbor Press.
Sternlicht, A. L. and Schultz, R. M. (1981). Biochemical studies of mammalian oogenesis: kinetics of accumulation of total and poly(A)-containing RNA during growth of the mouse oocyte. J. Exp. Zool. 215,191 -200.[CrossRef][Medline]
Sun, Q. Y. and Schatten, H. (2006). Regulation
of dynamic events by microfilaments during oocyte maturation and
fertilization. Reproduction
131,193
-205.
Tong, Z. B., Gold, L., De Pol, A., Vanevski, K., Dorward, H.,
Sena, P., Palumbo, C., Bondy, C. A. and Nelson, L. M. (2004).
Developmental expression and subcellular localization of mouse MATER, an
oocyte-specific protein essential for early development.
Endocrinology 145,1427
-1434.
Wang, Q. and Latham, K. E. (1997). Requirement for protein synthesis during embryonic genome activation in mice. Mol. Reprod. Dev. 47,265 -270.[CrossRef][Medline]
Wassarman, P. M. and Josefowicz, W. J. (1978). Oocyte development in the mouse: an ultrastructural comparison of oocytes isolated at various stages of growth and meiotic competence. J. Morphol. 156,209 -235.[CrossRef][Medline]
Worrad, D. M., Turner, B. M. and Schultz, R. M. (1995). Temporally restricted spatial localization of acetylated isoforms of histone H4 and RNA polymerase II in the 2-cell mouse embryo. Development 121,2949 -2959.[Abstract]
Wright, P. W., Bolling, L. C., Calvert, M. E., Sarmento, O. F., Berkeley, E. V., Shea, M. C., Hao, Z., Jayes, F. C., Bush, L. A., Shetty, J. et al. (2003). ePAD, an oocyte and early embryo-abundant peptidylarginine deiminase-like protein that localizes to egg cytoplasmic sheets. Dev. Biol. 256,73 -88.[Medline]
Wu, X., Viveiros, M. M., Eppig, J. J., Bai, Y., Fitzpatrick, S. L. and Matzuk, M. M. (2003). Zygote arrest 1 (Zar1) is a novel maternal-effect gene critical for the oocyte-to-embryo transition. Nat. Genet. 33,187 -191.[CrossRef][Medline]
Zamboni, L. (1970). Ultrastructure of mammalian oocytes and ova. Biol. Reprod. 2 Suppl., 44-63.[CrossRef][Medline]
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