Wellcome Trust/Cancer Research UK Institute, and Department of Genetics,
University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
* On leave from the Department of Experimental Embryology, Polish Academy of
Sciences, Jastrzebiec, Poland
Author for correspondence (e-mail:
mzg{at}mole.bio.cam.ac.uk)
Accepted 12 September 2002
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SUMMARY |
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Key words: Sperm, Egg, Polarity, Cleavage, Blastocyst pattern, mouse
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INTRODUCTION |
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However, we have recently learnt that early patterning of the mouse embryo
in normal, unperturbed development relates to the polarity established at the
very beginning of embryonic life. It appears that the first cleavage division
separates the mouse zygote into two halves that have a bias to follow
distinguishable fates (Piotrowska and
Zernicka-Goetz, 2001;
Piotrowska et al., 2001
;
Gardner, 2001
). Specifically,
one of the 2-cell embryo blastomeres cleaves ahead of its sister and tends to
contribute most of its descendants to the embryonic part of the blastocyst,
whereas the other, later dividing one, contributes progeny predominantly to
the abembryonic part (Piotrowska et al.,
2001
). The plane of this first cleavage division appears to relate
not only to the site of the previous meiotic division marked by the
second polar body (Plusa et al.,
2002a
) but also to the position of the fertilisation cone that
emerges at the site where the sperm enters the egg
(Piotrowska and Zernicka-Goetz,
2001
; Plusa et al.,
2002b
). The position of sperm penetration also correlates with the
division asynchrony between 2-cell embryo blastomeres
(Bennett, 1982
;
Piotrowska and Zernicka-Goetz,
2001
). This is shown by the observation that the first blastomere
to divide from the 2-cell to the 4-cell stage is generally the one that
acquires the part of the zygote cortex at which the sperm entered the egg.
Accordingly, these findings give rise to the hypothesis that in normal
development of an embryo the act of fertilisation itself might contribute to
setting up embryonic patterning
(Piotrowska and Zernicka-Goetz,
2001
; Piotrowska et al.,
2001
), as demonstrated in so many different species (see
Goldstein and Hird, 1996
;
Sawada and Schatten, 1989
;
Sardet et al., 1989
;
Roegiers et al., 1999
;
Vincent and Gerhart,
1987
).
The role of the sperm in the early patterning of the mouse embryo has been
questioned by Davis and Gardner (Davis and
Gardner, 2002) who have monitored the position of sperm components
taken up into the egg cytoplasm, namely the anterior part of the sperm tail
and the sperm-derived mitochondria, and related these to the first cleavage.
Unlike the earlier studies, they did not however mark the position of the
fertilisation cone, the egg's immediate cytoskeletal response to sperm
penetration, and this may help explain the discrepancy between the
interpretation of data from the different groups. In addition to these two
points of view it is possible that the oocyte itself possesses an endogenous
polarity that is effectual only in the context of a role for the sperm.
These viewpoints could be evaluated by studying the early patterning in
eggs lacking a sperm parthenogenetic embryos. Parthenogenetically
activated mouse eggs can develop into blastocysts and some even to early
post-implantation stages, by which point they die owing to lack of expression
of certain paternally derived genes (Barton
et al., 1984; McGrath and
Solter, 1984
). A comparison of fertilised and parthenogenetically
activated embryos could provide further insight into roles that the sperm
might have. If sperm penetration contributes to a symmetry-breaking event, is
its impact achieved by providing a positional cue that polarises the embryo
and affects the ensuing cleavage pattern, or is it by influencing the
synchrony of early cleavage divisions? It can be argued that any initial
asynchrony of cleavage could be sufficient to explain differential blastomere
fate. This is because the first dividing blastomere might contribute a greater
proportion of smaller cells earlier and these would contribute to the
embryonic part as they would be preferentially enclosed by the bigger, later
dividing cells. Hence if embryo patterning arises solely from the asynchrony
in the second cleavage between 2-cell blastomeres, one can expect that
patterning should be normal in parthenogenetic embryos, where cleavage
divisions are also asynchronous. In such a case the first blastomere to divide
in parthenogenetic embryos would also be expected to contribute preferentially
to the embryonic part, as in zygotes. Alternatively, if sperm entry provides a
positional cue that polarises the embryo, the early patterning of
parthenogenetic and fertilised embryos should differ.
To address whether 2-cell blastomeres have a tendency to follow distinguishable (embryonic and abembryonic) fates without any reference to the event of fertilisation, we have now studied development of early patterning in parthenogenetic eggs. To this end we used three different methods to activate eggs parthenogenetically one that yields haploid eggs and two others that yield diploid eggs. Our studies reveal that both the spatial contributions of progeny of 2-cell blastomeres to the blastocyst and the consequences of asynchrony in the early cleavage divisions differ between fertilised and parthenogenetic embryos. These findings are supported by further experiments to examine the order of division and developing spatial pattern in fertilised eggs in which the cortical cytoplasm at the site of sperm penetration has been removed. Together these results implicate a role for the sperm in both of these processes and indicate that although cleavage asynchrony contributes to assigning cells to specific blastocyst regions, on its own it appears insufficient to define the blastomeres' fate.
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MATERIALS AND METHODS |
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Such activated or fertilised eggs were transferred into KSOM medium supplemented with amino acids (KSOM+AA) and with 4 mg/ml of BSA (Speciality Media, Inc. Lavallette, NJ) and cultured in 5% CO2 and at 37°C to the 2-cell stage when their blastomeres were labelled. Embryos were observed under an inverted (Leica) microscope using DIC optics and micromanipulated with Leica micromanipulators using a De Fonbrune suction-force pump.
Micromanipulation
The additional series of experiments was designed to assess the role of the
position at which sperm entered the egg. Zygotes were recovered shortly after
fertilisation and their fertilisation cones were labelled with a fluorescent
bead as described above. Such eggs were cultured further, until the male
pronucleus had migrated towards the egg centre (approximately 3 hours after
fertilisation), at which stage the region of the egg that was marked by the
bead was removed by micromanipulation using techniques similar to those
described by McGrath and Solter (McGrath
and Solter, 1983). This operation removed approximately 13% of the
egg volume. The site from which the bead marking the sperm entry was removed
was relabelled by attaching another fluorescent bead. In a group of control
eggs another region of the cortex of the zygote (approximately 90° from
the visible fertilisation cone) was removed instead.
Labelling of blastomeres
DiI, DiD or DiO (Molecular Probes) was dissolved in virgin olive oil at
60°C, allowed to cool and then used immediately. Labelling was
accomplished by pressing the tip of the injection needle against the
blastomere membrane avoiding its penetration, then expelling a microdroplet
against the membrane, which absorbed the dye. Embryos with blastomeres
labelled with dyes of different colours were subsequently cultured in KSOM+AA
medium in 5% CO2 and at 37°C. They were first observed every
30-40 minutes during their 2-to 4-cell stage transition to evaluate the order
of blastomere division and finally at the expanding blastocyst stage when they
were analysed by confocal microscopy.
Analysis
Confocal analysis of blastocysts was performed on live embryos. Blastocysts
were observed by taking optical sections every 7 µm. By examining all
sections in each series, it was possible to determine the distribution of
labelled cells into the embryonic part (a part including the polar
trophectoderm and `deeper' cells of the inner cell mass ICM),
abembryonic part (a part including mural trophectoderm) and a boundary zone
between them. The boundary zone between these two parts was defined as a layer
approximately one cell deep and parallel to the blastocoelic surface of the
ICM as suggested in a previously described model
(Piotrowska and Zernicka-Goetz,
2001). In the first analysis, blastocysts were scored depending
upon the degree to which predominantly embryonic or abembryonic clones
extended beyond this boundary zone according to the criteria defined in the
legend to Fig. 2. The angle
between the clonal border and the boundary zone was defined by examining a
series of eight to ten confocal sections for each parthenogenetic blastocyst
to evaluate both the position of the clonal borders and the blastocoel, which
we drew as a line at the mid-points between cell boundaries, and tangential to
the cavity respectively.
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In a separate analysis of the distribution of descendants derived from the first versus second 2-cell blastomere to divide, we scored the cellular composition of all three parts of the blastocyst: embryonic, abembryonic and the boundary zone. Owing to the uneven labelling of cell membranes with dyes used here and lack of definition of the boundaries between the cells, it was difficult to obtain precise counts of cell numbers of intact embryos, therefore these counts should be regarded only as estimates. To be able to calculate a total number of cells derived from each (early and late dividing) blastomere in the whole blastocyst, we dissociated each of the embryos into individual cells and counted them. To this end, after confocal microscopy, we briefly exposed the blastocysts to acid Tyrode's solution to remove the zona pellucida and then we treated them with 0.5% trypsin (in Hank's buffered saline with 0.04% EDTA) for 5 minutes at 37°C before dispersing them into individual cells using thorough pipetting. Each cell in the blastocyst was either completely or substantially labelled by one of the two dyes.
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RESULTS |
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To follow the fate of descendants derived from 2-cell blastomeres and their
contribution to specific parts of the blastocyst we marked them with dyes of
different colours (Piotrowska et al.,
2001). The distribution of the two types of progeny was analysed
by dividing the blastocysts into 3 parts: embryonic, abembryonic and a
boundary zone between them defined as a layer approximately one cell deep and
parallel to the blastocoelic surface of the ICM, as previously described
(Piotrowska et al., 2001
). We
refer to regions of the blastocyst lying on either side of this boundary zone
as the embryonic or abembryonic parts, according to whether they include ICM
or the blastocoel, respectively. Two types of analysis enabled direct
comparisons to be made with the distribution of progeny of blastomeres in
fertilised embryos (Piotrowska et al.,
2001
). The first excluded cells lying in the boundary zone and
assessed the extent to which clones derived from each 2-cell blastomere
extended beyond it. The second analysis focused on the specific contribution
of descendants of the first versus the second 2-cell blastomere to divide into
each of the three parts of the blastocyst: embryonic, abembryonic and the
boundary zone. We also evaluated the relationship between the clonal border
(the interface between descendants of the 2-cell blastomeres, a reflection of
the first cleavage plane) and the boundary zone (the morphological division of
the blastocyst into its embryonic and abembryonic parts) for each embryo.
Parthenogenetic eggs differ from fertilised eggs in the fate of their
2-cell blastomeres
In the first analysis of the distribution of the progeny of 2-cell
parthenogenetic blastomeres, blastocysts were classified into four categories
depending upon the extent to which they conformed to the expectation (based on
zygotes) (Piotrowska et al.,
2001) that the embryonic part would be derived predominantly from
one 2-cell blastomere and the abembryonic part from the other. The progeny of
each 2-cell blastomere were scored separately according to the number of its
descendants that instead of being localised in either the embryonic or
abembryonic part, had come to lie in the opposite part of the blastocyst,
beyond the boundary zone. Blastocysts in which the 2-cell blastomere progeny
occupied predominantly either the embryonic or abembryonic part, thus having
only 3 or fewer cells of the clone (up to approximately 10% of the total cell
number at this stage) lying beyond the designated boundary zone, were scored
as either ++ (0-2 cells beyond) or + (3 cells beyond). If more than 3 cells
were found on the other side of the boundary zone, blastocysts were scored as
either (4 cells beyond) or (5 or more cells beyond)
(Fig. 2A-H).
Haploid parthenogenetic eggs
We found that the pattern of blastocysts in haploid parthenogenotes was
very variable. Thus, only 31% (8/26) and 39% (10/26) of blastocysts had clones
that occupied predominantly the embryonic or abembryonic parts respectively
(++ and + categories in Fig.
2A,B). This contrasts with 85% and 72% in the comparable
categories in the case of fertilised embryos
[Fig. 2G-H, data from
Piotrowska et al. (Piotrowska et al.,
2001)]. Half (54%) of haploid parthenogenetic embryos, compared to
6% in zygotes, fell into the -- category in which the spatial orientation of
the border between 2-cell blastomere clones departs dramatically from the
boundary zone (Fig. 2, another
example of this distribution is shown in
Fig. 3B). Thus in contrast to
fertilised eggs, the majority of progeny of each 2-cell blastomere in haploid
parthenogenotes tended not to lie exclusively in either the embryonic or
abembryonic parts of the blastocyst, but was distributed throughout the
embryonic-abembryonic axis. When we measured the angle between the clonal
border and the embryonic-abembryonic boundary in parthenogenetic blastocysts
we also found it to be much greater (59°±25°,
Table 1 and
46°±38°, Table
2; also see below) in comparison with that of fertilised embryos
(26°±19°) (Piotrowska et
al., 2001
). Thus, this second assessment confirms that the
position of the border of clones derived from each 2-cell blastomere bears no
relationship to the embryonic-abembryonic axis in the majority of haploid
parthenogenetic blastocysts.
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Diploid parthenogenetic eggs
The spatial pattern of diploid parthenogenetic blastocysts also
substantially differed from the pattern that developed after fertilisation.
Specifically, in the group of embryos generated by activating the oocytes and
then treating them with cytochalasin to prevent the extrusion of the second
polar body (2n CD; Fig.
1), only a minority of blastocysts were scored within the ++ and +
categories: 33% (11/33) and 24% (8/33) of blastocysts, depending whether
clones occupied predominantly the embryonic or abembryonic part respectively
(Fig. 2C,D). In this group of
parthenogenotes, 42% (14/33) were scored in the - - category (example shown in
Fig. 3C). The angle between the
clonal border and the embryonic-abembryonic boundary was also greater than in
fertilised eggs (Tables 1 and
2).
In diploid parthenogenetic embryos that were allowed to extrude then made to regain their second polar bodies (2n PB, Fig. 1), 54% (18/33) or 48% (16/33) of blastocysts were scored within the ++ and + categories, depending whether clones occupied predominantly the embryonic or abembryonic part respectively (Fig. 2E,F). In this group of activated embryos 24% (8/33) were scored in the - - category and thus showed a deviation between the spatial orientation of the clonal border and the embryonic-abembryonic boundary (example shown in Fig. 3D). There was also a substantial angle between the clonal border and the embryonic-abembryonic boundary in this group of embryos (Tables 1 and 2).
Thus, the clonal border between 2-cell blastomeres progeny in haploid and
diploid parthenogenetic embryos did not tend to predict the boundary between
embryonic and abembryonic parts of the blastocyst in the majority of embryos
as it did in fertilised eggs. The most extreme difference in distribution of
2-cell blastomere progeny between fertilised and parthenogenetic embryos was
seen in the haploid parthenogenotes and the least difference was seen in
diploid parthenogenotes generated by electrofusion. Nevertheless, the
distribution of embryos into analysed categories (++ and +, -, and - -) was
significantly different between fertilised eggs and each of the three types of
parthenogenetically activated eggs (P<0.001, 2
test 2d.f. in each case). When blastocyst patterning was assessed by measuring
the variation in the tilt between the clonal border and the boundary zone it
was found to be random in parthenogenotes, while in fertilised embryos it
showed a non-random distribution
(Piotrowska et al., 2001
).
Together these results indicate that the orientation of embryonic-abembryonic
axial polarity is not solely an intrinsic property of the egg itself and
indicates that fertilisation plays a role in establishing it.
Parthenogenetic eggs differ from fertilised eggs in that the first
2-cell blastomere to divide does not necessarily contribute a majority of
cells to the blastocyst
The differences in behaviour of clones in parthenogenetic embryos versus
fertilised ones was further clarified by determining the number of cells in
the blastocyst descended from each 2-cell blastomere. In fertilised eggs one
blastomere cleaves ahead of the other at the 2- to 4-cell stage transition.
The first dividing blastomere contributes more cells to the blastocyst
(Piotrowska et al., 2001).
This is not necessarily a result of the persistence of shorter subsequent cell
cycles in this lineage and may be accounted for solely by the earlier division
at the first cycle (Kelly et al.,
1978
; Graham and Deussen,
1978
). Because in fertilised embryos it is the progeny of the
2-cell blastomere inheriting the sperm entry position that have been shown to
maintain a division advantage, we wondered whether a similar tendency to
divide earlier would be maintained in parthenogenetic blastomeres.
To assess this we analysed the number of cells in the blastocyst derived
from the first and the second blastomere to divide in all three groups of
parthenogenotes (Fig. 3E,F). We
found that while in the majority (76%, 37/49)
(Piotrowska et al., 2001) of
fertilised embryos the 2-cell blastomere that divides earlier contributes more
cells to the blastocyst, this was not the case for parthenogenetic embryos.
Only in half (54%, 14/26) of haploid parthenogenotes did the first 2-cell
blastomere to divide contribute more cells to the blastocyst
(Table 1). In the remaining
embryos, the 2-cell blastomere dividing first contributed either an equal
number or fewer cells (Table
2). A similar conclusion was reached following the analysis of
diploid parthenogenetic eggs generated by cytochalasin treatment. Again only
half (55%, 13/24) of embryos showed the first 2-cell blastomere to divide to
contribute more blastocyst cells (Tables
1 and
2). However diploid
parthenogenetic embryos in which the second polar body had been electrofused
back into the egg appeared to behave more like fertilised embryos in this
respect. In this group the proportion of embryos in which the first blastomere
to divide made a greater contribution to the blastocyst was higher (67%,
22/33) than in the two other groups of parthenogenetic embryos
(Table 1 and
Table 2). Thus fertilisation
appears to have an effect on the timing of cell division that to some extent
might be mimicked by the experimental manipulations to generate
parthenogenetic embryos through electrofusion.
To confirm that fertilised and parthenogenetic embryos differ in their
ability to maintain the order of their blastomere divisions, we followed the
first two cleavages in two such groups. In fertilised embryos, we first marked
the fertilisation cone (appearing at the site of sperm entry) with fluorescent
beads. In agreement with our previous observations
(Piotrowska and Zernicka-Goetz,
2001), we found that the bead marked the first dividing blastomere
in 47% of 30 analysed embryos (group A), was found between the two blastomeres
in 26.5% of embryos (group B), and was associated with the later dividing
blastomere in the 26.5% of embryos (group C). After labelling 2-cell
blastomeres with dyes we found that in the great majority of embryos from
group A (93%, 13/14), the first dividing 2-cell blastomere again divided
first, up to the 5-cell stage. In 62.5% of embryos from group B the first
blastomere to divide again divided earlier, while in group C, there was an
equal chance of first and second blastomere to divide to enter the subsequent
division before their sisters. In the case of parthenogenotes (1n)
only in 60% (12/20) of embryos did the 2-cell blastomere that divided first
also divide first in the next cleavage division (to the 5-cell stage). When
the same group of embryos was analysed at the 8-cell stage both 2-cell
blastomeres had contributed an equal number of cells in 85% (17/20) of cases.
In 10% (2/20) of the remaining embryos, 5 cells were derived from the
earlier-dividing blastomere and in 5% (1/20), 5 cells were derived from the
later dividing one. Thus, both sets of observations lead to the same
conclusion: that while in fertilised eggs there is a strong tendency for at
least some of the progeny of the first blastomere to divide to maintain their
division advantage this is not the case in eggs not penetrated by sperm.
The order of division alone appears insufficient to establish the
fate of the 2-cell blastomeres
Our studies showed that parthenogenetic eggs differ from fertilised eggs in
that the discrepancy in division times between the 2-cell blastomeres progeny
was not preserved during development. Thus, we wondered whether this could
account for differences in embryonic pattern. If so this would mean that the
patterning develops exclusively on the basis of cleavage division order. A way
to test this possibility was for us to focus our analysis on the subset of
activated embryos that behaved more like fertilised eggs, in that the first
blastomere to divide contributed more cells to the blastocyst. We then asked
if this subset behaved similarly to fertilised eggs in showing a strong
preferential contribution of the first dividing 2-cell blastomere to the
embryonic part (Table 1 and
Tables 1A, 2A and 3A in Supplemental data:
http://dev.biologists.org/supplemental/).
We found that it did not in any of the three groups of parthenogenetic
eggs.
Our earlier study of fertilised eggs included an analysis of both early and
late blastocysts (Piotrowska et al.,
2001). If those data are reanalysed, focusing only on fertilised
embryos in which the first blastomere to divide contributed a majority of the
blastocyst cells, we find that on average 86% of the embryonic part is derived
from this cell. This value differs statistically from the corresponding value
for all three groups of parthenogenetic embryos (t-test,
P<0.001). Specifically, in the group of haploid parthenogenotes,
on average only 64% of the embryonic part consisted of the progeny of the
first blastomere to divide (Table
1 and Table 1A in Supplemental data:
http://dev.biologists.org/supplemental/).
In the same group of embryos, on average only 52% of the abembryonic part
consisted of progeny of the second blastomere to divide. Most noticeably,
there was no single embryo in which the first blastomere to divide made an
exclusive or nearly exclusive (
90%) contribution to the embryonic part
(Table 1A in Supplemental data:
http://dev.biologists.org/supplemental/).
This was in contrast to fertilised eggs, in which in a substantial proportion
of embryos the first blastomere to divide made an exclusive (6/49) or
90%
(14/49) contribution to the embryonic part
(Piotrowska et al., 2001
). In
those parthenogenetic embryos in which the second blastomere to divide
contributed more blastocyst cells, slightly more than half of the embryonic
part (57%) consisted of its descendants. In this group there was only one
embryo (1/12) in which the second blastomere to divide made a nearly exclusive
contribution to the embryonic part (Table 1B in Supplemental data:
http://dev.biologists.org/supplemental/).
This outcome was similar whether the parthenogenetic embryos were haploid
or diploid. In the subset of diploid parthenogenotes treated with cytochalasin
in which the first blastomere to divide contributed more cells, it contributed
on average 67% of the embryonic part (Table
1 and Table 2A in Supplemental data:
http://dev.biologists.org/supplemental/).
In the other subset, in which the second blastomere to divide contributed more
or an equal number of cells, it contributed 44% of the embryonic part
(Table 2 and Table 2B in
Supplemental data:
http://dev.biologists.org/supplemental/).
Similarly in a comparable subset of diploid parthenogenetic embryos generated
by electrofusion, an average of 66% of the embryonic part was occupied by the
progeny of the first blastomeres to divide when they contributed more cells to
the blastocysts (Table 1 and
Table 3A in Supplemental data:
http://dev.biologists.org/supplemental/).
Moreover, no single embryo in this group showed an exclusive (and only one
90%) contribution of early dividing descendants to the embryonic part.
Taken together these results indicate that unlike fertilised eggs, there is not a strong tendency for the 2-cell blastomeres of parthenogenetic eggs to follow embryonic or abembryonic fates. Moreover, this does not appear to be attributable to an exclusive role for sperm in influencing the timing of the second cleavage because even when the progeny of the first parthenogenetic blastomere to divide maintain a division advantage, they do not show such a strong predisposition to occupy the embryonic part of the blastocyst, as observed in fertilised eggs.
Removal of the cortical cytoplasm associated with the position of
sperm entry in zygotes disturbs spatial patterning of the blastocyst
The above experiments indicated that fertilisation provides a bias in
establishing the fate of blastomeres that is not achieved solely by an effect
on the timing of cell division. Fertilisation of the mouse oocyte results in
at least 3 events: introduction of the male set of chromosomes and other sperm
components into the egg, global egg activation as well as localised changes at
the site of the sperm entry. Because previous experiments observed a
correlation between the position of the fertilisation cone at the site of
sperm entry and the pattern of cleavage
(Piotrowska and Zernicka-Goetz,
2001; Plusa et al.,
2002b
), we decided to test directly the importance of localised
events imposed upon the blastocyst axial organisation by sperm penetration.
With this aim we surgically removed cortical cytoplasm either at the site of
sperm entry (Fig. 4A-D) or, in
a control group of embryos, from elsewhere on the embryo surface
(Fig. 4E-H). At the 2-cell
stage we labelled blastomeres, observed their order of division to the 4-cell
stage, and then allowed the embryos to develop to the blastocyst.
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When we analysed the distribution of blastocyst cells between the embryonic and abembryonic parts with respect to the boundary zone, only 37% (10/27) of embryos in which cortical cytoplasm associated with sperm entry was removed fell into the ++ and + category when the embryonic part was analysed, and 52% (14/27) when the abembryonic part was considered. This compared with 75% (18/24) and 79% (19/24), respectively, of control manipulated embryos. When the sperm entry-associated cortex was removed, 30% (8/27) of blastocysts scored in the - - category, compared with only 4% (1/24) in the control group. Thus while the control manipulated eggs are similar to non-manipulated fertilised eggs, in that descendants of the 2-cell blastomeres tend to occupy either the embryonic or abembryonic parts, this tendency is lost following surgical removal of cortical cytoplasm around the site of sperm entry.
An analysis of the timing of the division of the 2-cell blastomeres'
progeny led to a similar conclusion. Out of 27 blastocysts developed from eggs
with the sperm entry cortical cytoplasm removed, only half (14/27) had a
greater number of cells derived from the first dividing blastomere (Tables
1 and
2; Table 4A and 4B in
Supplemental data:
http://dev.biologists.org/supplemental/).
Also there was no tendency of this first 2-cell blastomere to divide to
contribute the majority of the embryonic part. Thus on average only 53% of the
embryonic part was derived from the first blastomere to divide when this
contributed a greater proportion of the blastocyst cells and 51% when it
contributed a lesser portion (Tables
1 and
2). These results indicate that
in fertilised embryos lacking cortical cytoplasm around the sperm entry there
was a roughly equal possibility of the embryonic part being developed from
either 2-cell blastomere. By contrast, when a sector of cortical cytoplasm was
removed from the sperm entry site, the tendency for the first dividing 2-cell
blastomere to contribute more cells to the blastocyst remained. Although not
as dramatic as in non-manipulated embryos it was still seen in 71% (17/24) of
blastocysts (Tables 1 and
2). In those control
manipulated blastocysts in which there were more cells derived from the first
dividing 2-cell blastomere, it contributed on average 84% of the embryonic
part (Table 1; Table 5A in
Supplemental data:
http://dev.biologists.org/supplemental/).
This figure is similar to a contribution of 86% of the embryonic part by the
first dividing blastomere in non-manipulated fertilised embryos
(Piotrowska et al., 2001).
Moreover, within this control group, the earlier dividing 2-cell blastomere
made an exclusive or nearly exclusive contribution to the embryonic part in
47% (8/17) of embryos (Table 5A, in Supplemental data:
http://dev.biologists.org/supplemental/).
This was not observed in the embryos with the sperm entry-associated cortical
cytoplasm removed. Thus despite the surgical manipulation of control eggs
there is relatively little loss of normal embryo patterning. We conclude that
the disruption of the cortical cytoplasm specifically in the region of sperm
penetration does indeed influence the pattern of blastocyst development.
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DISCUSSION |
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Because the plane of the first cleavage division correlates with both the
position of the previous meiotic division the animal pole
(Plusa et al., 2002a) and the
position of the fertilisation cone that marks the sperm entry
(Piotrowska and Zernicka-Goetz,
2001
; Plusa et al.,
2002b
), the question arises of the extent of the role of the
oocyte, and that of the sperm in setting up early embryo patterning. It could
be argued that allocation of the progeny of 2-cell blastomeres in diploid
parthenogenetic blastocysts differs from that in normal embryos because their
animal pole has been perturbed by either cytochalasin treatment or
electrofusion. This could disturb factor(s) at the animal pole that influence
the first cleavage orientation. For this reason we also examined two
experimental situations in which the animal pole was not perturbed. But here
again we found that development of embryonic-abembryonic patterning in
relation to the first cleavage was lost. The first of these was in haploid
parthenogenetic eggs. In this case, the second meiotic division was not
perturbed and the resulting polar body subsequently came to lie between the
2-cell blastomeres. Thus the first cleavage divided the egg along the
animal-vegetal axis as it did in fertilised eggs. Yet the two blastomeres did
not follow predominantly embryonic or abembryonic fates. This suggests that
the division of the embryo into its two distinct parts cannot exclusively
depend on the information provided by the animal pole alone. That the failure
of such embryos to develop normal polarity is related to the absence of
fertilisation rather than haploid development per se, is indicated by
experiments in which the cortical cytoplasm at the sperm entry position was
surgically removed from fertilised eggs. In such surgically manipulated eggs,
the first cleavage also continued to respect the spatial information provided
by the animal pole, but it no longer divided the zygote into blastomeres with
embryonic or abembryonic destinies. If cortical cytoplasm was removed in
control embryos at approximately 90° to the sperm entry site then the
first cleavage did tend to divide the egg into these two blastocyst parts.
These experimental situations indicate that when sperm does not participate in
development, the tendency for the embryo to be partitioned into embryonic and
abembryonic parts at the first cleavage is lost.
Can we determine whether sperm polarises the embryo directly or exerts its effect through the order of cell division? Our findings that the patterning of the fertilised and parthenogenetic embryos are different and yet both show asynchronous early divisions seem at first sight to indicate that the sperm penetration may have a directly polarising effect. However, we have unexpectedly observed that, unlike fertilised eggs, the progeny of the first blastomere to divide in parthenogenetic eggs show no tendency to retain their division advantage. The one exceptional group in this respect are parthenogenetic eggs subjected to electrofusion. At present we cannot conclude whether the ability of one 2-cell blastomere to retain a division advantage in this group of eggs relates to a response to the electric field or the process of fusion that is mimicking some aspect of sperm penetration. Regardless of the explanation, this `electrofused' group of parthenogenotes is similar to the other groups in showing no strong tendency for progeny of the first dividing 2-cell blastomere to occupy predominantly the embryonic part of the blastocyst as occurs in fertilised eggs. This is despite the net difference in number of progeny derived from the early dividing versus later dividing 2-cell blastomere in parthenogenetic embryos being often similar to fertilised ones. This therefore suggests that the order of cleavage itself might not be the sole factor responsible for assigning cells to the embryonic and abembryonic parts. It does not however preclude the possibility that in fertilised eggs the sperm might contribute to defining embryonic pattern by influencing the order of cleavage.
Taken together, our results provide a strong support for previous findings
that fertilising sperm has a role in early patterning of the embryo in normal,
unperturbed development (Piotrowska and
Zernicka-Goetz, 2001; Plusa et
al., 2002b
). They do not support the notion of Davis and Gardner
(Davis and Gardner, 2002
) who
concluded that since they could not detect a relationship between the
localisation of sperm components within the egg and the first cleavage plane,
fertilisation plays no role. Davis and Gardner
(Davis and Gardner, 2002
) also
criticised the use of lectins to mark the region of the fertilisation cone in
previous experiments, arguing that this method should preferentially mark the
zona pellucida. However, they overlooked the observations that even
zona-denuded eggs could be marked in this way and the marker still remained
close to the cleavage plane in the majority of embryos
(Plusa et al., 2002b
). Their
references to the Concanavalin A and phytohaemagglutinin binding properties of
the zona pellucida and the egg surface (excluding the fertilisation cone) do
not discredit the observation that the beads remained bound to the plasma
membrane. While the precise mechanism for bead attachment is difficult to
ascertain, it may have been due to initially weak affinities that are at later
stages enhanced by recruitment of lectin-binding receptors from adjacent
membrane. Davis and Gardner (Davis and
Gardner, 2002
) were also inaccurate in their criticism of control
experiments to ensure that surface markers maintained their relative
positions. Contrary to their assertion, a control with microspheres placed
randomly at the equator of the egg as well as adjacent to, or diametrically
opposite, the second polar body was performed
(Piotrowska and Zernicka-Goetz,
2001
). Furthermore, the validity of such markers was demonstrated
by Plusa et al. (Plusa et al.,
2002b
) through the use of double labelling techniques to mark
independent sites and thereby show that only the bead attached to the
fertilisation cone and not one attached to a random position, tends to mark
the first cleavage plane. The possibility cannot be ruled out that pressure
placed on the fertilisation cone by positioning the marker bead could itself
have influenced the plane of cleavage. Nevertheless, beads positioned in this
way did come to lie on the boundary between the embryonic and abembryonic
parts in the majority of blastocysts suggesting that 2-cell blastomeres have a
tendency to follow distinguishable fates, a finding confirmed by lineage
tracing studies (Piotrowska and
Zernicka-Goetz, 2001
;
Piotrowska et al., 2001
).
Perhaps then the differences between the conclusions reached by ourselves and
Davis and Gardner lie in different parameters that were scored by the two
groups; cortical events and the fertilisation cone, on the one hand and
internal events, localisation of sperm components taken up by the egg, on the
other.
Where then does the `cue' provided by sperm entry act? Since the
partitioning of the embryo into its future embryonic and abembryonic parts by
the first cleavage can be disturbed by changes in the cortical cytoplasm
associated with the site of sperm entry, it can be concluded that the cue
provided by sperm does not reside exclusively within, nor is it directly
associated with, the male pronucleus. However, the relative positioning of the
male and female pronuclei may be important for spindle orientation as their
two sets of chromosomes do not mix for the first division cycle
(Mayer et al., 2000). If this
were the case, it is possible that a potential secondary effect of
micromanipulation could be to disrupt the position of the male pronucleus in
relation to the female counterpart and thereby disrupt patterning. We are
unable to exclude this possibility. However as manipulating even control
embryos could interfere with the position of male and female pronuclei, it
seems more likely that the disruption of patterning following removal of the
fertilisation cone site reflects rather the importance of the localised events
at this site. Our study does not allow us to distinguish whether it is a
response of the egg at the site of sperm entry itself or a local concentration
of specific sperm components (Hewitson et
al., 2002
) that is important for early patterning. Either of these
in turn might be reflected in the changes of organisation of the egg cortex we
see after sperm entry that may be in some way analogous to the rearrangements
of the cortex of the Xenopus egg that occur upon sperm penetration
(Vincent and Gerhart, 1987
;
Gerhart, 1991).
Could it be that besides the information provided by the animal pole to orient the first cleavage, the egg has an additional role in establishing embryo polarity? We note that there is some patterning, although significantly reduced, in diploid parthenogenetic embryos that have been subjected to electrofusion. Does this mean that the egg has some inherent patterning and if so is it partially activated by electrofusion as it would be by fertilisation? We certainly take into account this possibility and at present cannot determine the extent to which the role of the sperm is to activate pre-existing elements of polarity in addition to provide a `cue' that breaks the egg's symmetry.
It is difficult to be certain of the later developmental consequences of
the failure of the first cleavage to partition cells between embryonic and
abembryonic parts in parthenogenetically activated embryos. This is because
such embryos normally die shortly after implantation because of lack of
expression from the paternally derived genes
(McGrath and Solter, 1984;
Barton et al., 1984
). However,
given that when a male pronucleus is transplanted to a parthenogenetic egg the
resulting embryo can develop to term (Mann
and Lovell-Badge, 1984
) the early patterning events we describe
here are unlikely to be essential for further development. Indeed, the mouse
embryo is highly regulative in its development and thus there are likely to be
means of countering spatial perturbations imposed at early developmental
stages. Hopefully an analysis of the re-establishment of patterning following
its perturbation should give us valuable clues to the mechanisms that operate
in normal egg development.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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Barton, S. C., Surani, M. A. and Norris, M. L. (1984). Role of paternal and maternal genomes in mouse development. Nature 311,374 -376.[Medline]
Bennett, J. (1982). Sperm entry point is related to early division of mouse blastomeres. J. Cell Biol. 95,163a .
Davis, T. J. and Gardner, R. L. (2002). The
plane of first cleavage is not related to the distribution of sperm components
in the mouse. Hum. Reprod.
17,2368
-2379.
Gardner, R. L. (2001). Specification of
embryonic axes begins before cleavage in normal mouse development.
Development 128,839
-847.
Gerhart, J., Doniach, T. and Stewart, R. (1991). In Gastrulation: Movements, Patterns and Molecules (ed. R. Keller, Clark, WH Griffin), pp.57 -76. New York: Plenum.
Goldstein, B. and Hird, S. N. (1996).
Specification of the anteroposterior axis in Caenorhabditis elegans.
Development 122,1467
-1474.
Graham, C. F. and Deussen, Z. A. (1978). Features of cell lineage in preimplantation mouse development. J. Embryol. Exp. Morphol. 48,53 -72.[Medline]
Gurdon, J. B. (1992). The generation of diversity and pattern in animal development. Cell 68,185 -199.[Medline]
Hewitson, L., Simerly, C. R. and Schatten, G. (2002). Fate of sperm components during assisted reproduction: implications for infertility. Hum. Fertil. 5, 110-116.
Kelly, S. J., Mulnard, J. G. and Graham, C. F. (1978). Cell division and cell allocation in early mouse development. J. Embryol. Exp. Morphol. 48, 37-51.[Medline]
Mann, J. R. and Lovell-Badge, R. H. (1984). Inviability of parthenogenones is determined by pronuclei, not egg cytoplasm. Nature 310,66 -67.[Medline]
Mayer, W., Smith, A., Fundele, R. and Haaf, T.
(2000). Spatial separtion of paternal genomes in preimplantation
mouse embryos. J. Cell Biol.
148,629
-634.
McGrath, J. and Solter, D. (1983). Nuclear transplantation in the mouse embryo by microsurgery and cell fusion. Science 220,1300 -1302.[Medline]
McGrath, J. and Solter, D. (1984). Completion of mouse embryogenesis requires both the maternal and paternal genomes. Cell 37,179 -183.[Medline]
Plusa, B., Grabarek, A., Piotrowska, K., Glover, D. M. and Zernicka-Goetz, M. (2002a). Site of the previous meiotic division directs the first cleavage plane of the mouse egg. Nature Cell Biol. 4,811 -815.[CrossRef][Medline]
Plusa, B., Piotrowska, K. and Zernicka-Goetz, M. (2002b). Sperm entry position provides a surface marker of the first cleavage plane of the mouse zygote. Genesis 32,193 -198.[CrossRef][Medline]
Piotrowska, K., Wianny, F., Pedersen, R. A. and Zernicka-Goetz,
M. (2001). Blastomeres arising from the first cleavage
division have distinguishable fates in normal mouse development.
Development 128,3739
-3748.
Piotrowska, K. and Zernicka-Goetz, M. (2001). Role for sperm in spatial patterning of the early mouse embryo. Nature 409,517 -521.[CrossRef][Medline]
Roegiers, F., Djediat, C., Dumollard, R., Rouviere, C. and
Sardet, C. (1999). Phases of cytoplasmic and cortical
reorganizations of the ascidian zygote between fertilization and first
division. Development
126,3101
-3117.
Sawada, T. and Schatten, G. (1989). Effects of cytoskeletal inhibitors on ooplasmic segregation and microtubule organization during fertilization and early development in the ascidian Molgula occidentalis. Dev. Biol. 132,331 -342.[Medline]
Sardet, C., Speksnijder, J., Inoue, S. and Jaffe, L. (1989). Fertilization and ooplasmic movements in the ascidian egg. Development 105,237 -249.[Abstract]
Vincent, J. P. and Gerhart, J. C. (1987). Subcortical rotation in Xenopus eggs: an early step in embryonic axis specification. Dev. Biol. 123,526 -539.[Medline]
Zernicka-Goetz, M. (2002). Patterning of the
embryo: the first spatial decisions in the life of a mouse.
Development 129,815
-829.