Human Reproduction Vol.22, No.3 pp. 798–806, 2007
doi:10.1093/humrep/del424
Advance Access publication November 24, 2006.
The axis of polarity of the mouse blastocyst is specified before
blastulation and independently of the zona pellucida
R.L.Gardner
Mammalian Development Laboratory, University of Oxford, Department of Zoology, South Parks Road, Oxford, UK
E-mail: [email protected]
BACKGROUND: Rather than being prepatterned, orientation of the embryonic–abembryonic (Em–Ab) axis of the
mouse blastocyst has been claimed to depend on the conceptus being constrained by its zona pellucida (ZP) during
blastulation. This hypothesis merited closer scrutiny, because it seemed at variance with observations on living
conceptuses. METHODS: Two-cell conceptuses with an oil drop injected into the lesser diameter (LD) of the ZP at
the first cleavage plane were cultured until shortly before blastulation when the blastomere underlying the drop was
labelled with carbocyanine dye. After removing the ZP, conceptuses were re-cultured to the blastocyst stage for
recording the position along the axis of the centres of the patches of labelled cells. RESULTS: These centres showed
significant bias towards the equatorial (Eq) region of the axis compared with those resulting from labelling a blastomere at random, even following softening of the ZP at the 2-cell stage. This was also true if conceptuses were
denuded at the 2-cell stage and the blastomere underlying an intact second polar body (PB) labelled in morulae.
CONCLUSIONS: These findings further support the view that the Em–Ab axis of the mouse blastocyst is normally
prepatterned and provide no evidence of a role for the ZP in its specification.
Key words: blastocyst/axis of polarity/prepatterning/zona pellucida
Introduction
Non-random mapping of features found in the zygote and 2-cell
conceptus on the blastocyst in the mouse has been found in
several studies using non-invasive or minimally invasive marking techniques (Gardner, 1997, 2001a; Piotrowska et al., 2001;
Fujimori et al., 2003; Gardner and Davies, 2006). It has been
argued elsewhere that such relationships, which include the conservation of bilateral symmetry (Gardner and Davies, 2006), are
difficult to explain without invoking prepatterning (Gardner,
2005, 2006). However, others have attributed them to topological constraints operating during cleavage (Hiiragi and Solter,
2004; Rossant and Tam, 2004).
Particularly pertinent to the case for prepatterning are findings
based on the use of two strategies that have the virtue of being
strictly non-invasive, because they avoid introducing any exogenous markers into either the cytoplasm or the membranes of
cells of the conceptus. The first was to record the location on the
surface of the blastocyst of the second polar body (PB) in the
nearly two-thirds of cases where it remained intact (Gardner,
1997). This body, which almost invariably lies between the two
blastomeres following first cleavage (Howlett and Bolton,
1985), mapped significantly more frequently to the equator than
to either polar region of blastocysts of closed-bred Pathology
Oxford (PO) albino mice. The preferential equatorial (Eq) location
of the second PB was particularly intriguing, because it was evidently still physically attached to the surface of the conceptus by
the intercellular bridge formed during its production (Gardner,
1997). If, as seems likely, the second PB is therefore not free to
move independently of its point of attachment to the surface of
the conceptus, its non-random location on the blastocyst implies
that the embryonic–abembryonic (Em–Ab) axis of the blastocyst
tends to be orthogonal to the plane of first cleavage in the mouse.
Use of the second non-invasive strategy, which entailed
injecting small drops of mineral oil into the zona pellucida
(ZP) to mark features of the 2-cell conceptus, offered further
support for the existence of prepatterning. Thus, oil drops
placed in the ZP over the first cleavage plane and the outer
extremity of one blastomere tended to map, respectively, to the
equator versus either polar region of blastocysts. Motosugi
et al. (2005) have attributed these findings to imposition of
mechanical constraint on the conceptus by the ZP rather than to
prepatterning. Thus, they claim to have demonstrated that the
Em–Ab axis is not specified until blastulation when its orientation depends on the shape of the investing ZP. Where the ZP is
ellipsoidal, it is held to constrain the nascent blastocyst, so that
the blastocoele forms in alignment with its greater diameter
(GD). Because, according to these authors, the GD of the ZP
accords with the outer extremity of the blastomeres at the 2-cell
stage, they claim that this ‘mechanical constraint’ hypothesis
readily accounts for how oil drops injected into different
regions of the ZP at this stage are distributed over the surface
of the blastocyst (see Figure 1 for details). However, as
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Polarity specified before blastocyst stage
with the hypothesis of Motosugi et al. (2005) unless the ZP is
much more commonly spherical in the hybrid conceptuses they
studied than in the PO strain that was used for the oil marking
experiments. In fact, while the ZP is ellipsoidal in the overwhelming majority of PO conceptuses, it is usually more nearly oblate
(disk-like shape) than prolate (rugby-ball-like shape). This distinction, which Motosugi et al. (2005) fail to make, is important
because rotation of the Em–Ab axis of the conceptus will only be
constrained by the ZP if it is prolate (Gardner, 2001a, 2006).
Hence, one interpretation of the studies employing the second
PB or oil drops in the ZP as markers is that there is a conserved
relationship between the 2-cell stage and blastocyst with specific
regions of the former normally giving rise to specific regions of
the latter. The other is that such a relationship is apparent rather
than real because the conceptus is free to rotate within its ZP but
may be constrained by this envelope with regard to where its blastocoele forms. However, if the second PB is free to move over the
surface of the conceptus despite its tether (cited as unpublished
observations in Motosugi et al., 2005 and Hiiragi and Solter,
2006), its non-random location on the surface of the blastocyst
must likewise be due to some as yet undefined constraint.
A difficulty in designing experiments to distinguish between
these two contrasting hypotheses regarding how the Em–Ab
axis of the blastocyst is specified arises from confusion as to
whether mechanical constraint imposed by the ZP actually
directs the initial orientation of the blastocyst’s Em–Ab axis or
merely its final one. Thus, according to Motosugi et al. (2005):
Figure 1. Typical distribution on the blastocyst of oil drops in the zona
pellucida (ZP) used to mark features of the 2-cell conceptus (A) in an
earlier study (Gardner, 2001a). Blastocysts in side view (B1 and B2)
with mapping to one or other pole of the two oil drops placed over the
outer extremity of one 2-cell blastomere. Blastocyst in polar view (C)
with the 1 and 3 oils corresponding to the greater diameter (GD) versus
lesser diameter (LD) of the ZP at the first cleavage plane parallel versus
orthogonal to its bilateral plane. (D1–D4) According to Motosugi et al.
(2005), the results of these oil drop marking experiments can be
explained by imposition of mechanical constraint on the conceptus by
the ZP during blastocyst formation. The conceptus is supposedly fixed
in orientation at the 2-cell stage with the plane of first cleavage orthogonal to the ZP’s greatest diameter (D1). However, it is held to rotate
freely thereafter so that, as indicated by the position of the cross in a
morula (D2) and a nascent blastocyst (D3), the parts underlying the different oil sites in the ZP change as cleavage progresses. Finally, with
formation of the blastocoele, the conceptus is once again constrained by
the ZP which results in its embryonic–abembryonic (Em–Ab) axis
becoming aligned with the ZP’s greatest diameter, and thus the consistent mapping of the three oil sites on the blastocyst (D4).
discussed at length elsewhere (Gardner, 2006), the ‘mechanical
constraint’ hypothesis not only is difficult to reconcile with various observations on living conceptuses but fails to account satisfactorily for findings in several other studies. Thus, Motosugi
et al. (2005) state that mechanical constraint will operate to orient
the blastocoele, and hence the Em–Ab axis, only in the 50–60% of
conceptuses whose ZP is ellipsoidal, and not in the remaining 40–
50% for which it is said to be approximately spherical. However,
in the original study (Gardner, 2001a), oil drops injected into the
ZP at one end of its lesser diameter (LD) over the first cleavage
plane mapped to the Eq third of the surface in the great majority of
blastocysts (100 of 119). These findings are difficult to reconcile
Our model of Em–Ab polarity formation in the preimplantation
embryo predicts that in essentially all embryos, mechanical compression exerted through the ZP as well as spatial constraints will induce
formation of Em–Ab axis perpendicular to the short axis of the ZP.
Subsequently, however, Hiiragi and Solter (2006) state rather
more vaguely that ‘. . . the model does predict that, irrespective
of the initial orientation, the eventual em-ab axis will conform to
the enforced longest diameter of the ZP’.
Nonetheless, the prepatterning and mechanical constraint
hypotheses make distinct predictions regarding where the outer
blastomere in the late morula underlying an oil drop marking
one end of the LD of the ZP at the first cleavage plane would
map on the blastocyst following removal of the ZP before blastulation. Thus, according to the former hypothesis, it should
still lie preferentially in the Eq region of the Em–Ab axis
whereas the expectation from the latter hypothesis is that its
location should be entirely random.
Differentiating between these two possibilities is important
for deciding whether there should be concern about the
increasing clinical application to human reproduction in vitro
of invasive procedures such as ICSI and PGD/ preimplantation
genetic screening (PGS), or whether the marked regulative
capacity of early conceptuses is adequate to negate any perturbation these might cause.
Materials and methods
Mice
Mice of the PO closed-bred albino strain were used throughout the
study, with stocks kept under two different lighting regimes. The dark
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R.L.Gardner
period was from 1900 to 0500 h for one stock and from 1100 to 2300 h
for the other. All matings, including those with vasectomized males,
depended on natural rather than induced ovulation, and 2-cell conceptuses were recovered by flushing the excised oviducts of females
killed on the day after detecting a vaginal plug. Hepes-buffered modified synthetic oviductal medium enriched with potassium (mKSOM)
medium was used for recovery, short-term storage at room temperature and manipulation of conceptuses, and the bicarbonate-buffered
variant of this medium (Gardner and Davies 2000) for their culture at
37.5°C in microdrops in bacteriological dishes under mineral oil in an
atmosphere of 5% CO2 in air. Rounding of the blastomeres of 2-cell
conceptuses was encouraged by incubating them for up to 15 min in
pre-equilibrated calcium-free ovum culture (OC) medium containing
0.02% EGTA (Gardner and Davies, 2000).
Manipulation of conceptuses
For marking sites in the ZP, one or more small drops of soya oil were
injected into it, as described elsewhere (Gardner, 2001a). The ZP was
eliminated by briefly exposing conceptuses to acidified Tyrode’s saline
(AT), and softened by culturing them for 1.75–2.00 h in mKSOMcontaining proteinase K (PK) at 10 μg/ml (Sigma P-2308: 44 units/mg).
Individual outer blastomeres of advanced morulae were labelled by
microinjecting them with a drop of 1,1′-dioctadecyl-3–3-3′-3′-tetramethylindocarbocyanine perchlorate (DiI, Molecular Probes Inc., Eugene,
Oregon, USA) in catering quality soya or olive oil. The DiI was prepared
as a saturated stock in absolute ethanol, which was diluted 5-fold with
the mineral oil and then mixed vigorously to produce a fine suspension
for injection via a fine-tipped pipette. Morulae with an intact or softened
ZP were held for injection by gentle suction on the tip of a pipette. For
those that had been denuded the microscope stage was cooled to ∼5°C
with a thermo-circulator and a round-tipped glass probe used both to orient and to immobilize them by lowering its tip onto their upper surface
so as to compress them slightly against the floor of the drop.
Scoring of conceptuses
For detecting changes in shape of the ZP, NIH Image J software was
used on digital images to measure the distance between the centres of
oil drops injected into the ZP over the outer extremity of both blastomeres of 2-cell conceptuses. Appropriately oriented digital images of
blastocysts obtained from 2-cell conceptuses with distinctive oil drop
marking of the two ends of the GD and LD of their ZP at the first cleavage
plane were used to determine the angle of departure of these diameters
from the blastocyst’s equator. Blastocysts developing from advanced
morulae with a blastomere labelled with DiI were oriented individually in hanging drops in a Puliv chamber (Gardner and Davies, 2000)
with their Em–Ab axis horizontal and rotated about this axis for optimal display of the labelled cells for digital photography. Depending
on the depth of the fluorescent patch, between 1 and 3, fluorescent
images were captured per blastocyst, followed by a single differential
interference contrast (DIC) image. The fluorescent images were then
superimposed on the DIC image using Adobe Photoshop 7.0 software,
so that the percentage distance from the Em pole of the centre of patch
formed by the labelled blastomere along the Em–Ab axis could be
determined (Figure 2). A map measurer was used to ascertain the
location of the centre of patches that extended round one or other of
the blastocyst’s poles. Taking the centre of an entire patch as an indicator of the relative position of the originally labelled blastomere can
be misleading in two respects. First, outer blastomeres can produce
inner cell mass (ICM) cells which, because the blastocoele forms
eccentrically, may be remote from their progeny in the trophectoderm.
Hence, all labelled cells in the ICM were discounted. Secondly, rather
than being confined to the injected blastomere, DiI can sometimes
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Figure 2. Photographs of blastocysts developed from morulae in
which an outer blastomere was labelled with DiI (A–D). As illustrated
in (A), the location on the embryonic–abembryonic (Em–Ab) axis of the
centre of the patches of fluorescence in trophectoderm in percentage
distance from the Em pole was calculated as 100[ab + 0.5(ac – ab)]/ad.
spread to its sister if the two are still joined by a patent intercellular
bridge (Goodall and Johnson, 1984; Gardner, 2002). Because it is usually only partial, this spread typically results in a mixture of strongly
and more weakly fluorescing cells. Hence, where DiI-positive cells in
the trophectoderm could obviously be assigned to one of two distinct
classes in their intensity of fluorescence, the more weakly staining
ones were excluded. It should also be noted that with time DiI is
cleared from the extremities of cells, so that the fluorescent signal tends
to become concentrated centrally around nuclei. Finally, for each
experimental and control series, the number of centres falling within
the Em polar, Eq and Ab polar thirds of the Em–Ab axis was recorded.
Statistical analysis
Student’s t-test was used for assessing differences in mean diameter of
the ZP and the G-test with William’s correction (Sokal and Rohlf,
1995) for ascertaining whether distributions of the centres fluorescent
patches along the Em–Ab axis of blastocysts were heterogeneous or
differed significantly from random.
Results
Shape of the ZP parallel to the first cleavage plane
Initially, a series of 63 unselected 2-cell conceptuses were oriented with one blastomere vertically above the other (Figure 3)
to ascertain the frequency with which the ZP was obviously
oval rather than circular in profile parallel to the plane of first
cleavage. Measurements confirmed that the cross-section of
the ZP in this orientation was typically oval with its GD
exceeding its LD by >5% (LD/GD ratio, mean ± SE 0.91 ±
0.0059) in 51 of the 63 specimens (81%). Next, in a further
series of 2-cell conceptuses, both ends of the GD and the LD of
the ZP at the first cleavage plane (Figure 3B) were marked,
respectively, with two versus one oil drops. The conceptuses
were then cultured, so that the orientation of the two diameters
of the ZP relative to the Eq plane of the resulting blastocysts could
be determined. The results, which are presented in Figure 4, show
that the LD line was within 19.5° of parallel to the equator in
Polarity specified before blastocyst stage
Figure 3. Same 2-cell conceptus on its side (A) and with one blastomere vertically above the other (B) to show the outline of the zona
pellucida (ZP) is conspicuously oval in the latter view. The tip of an
injection pipette in (B) indicates where an oil drop would be placed in
the ZP to mark its lesser diameter (LD) at the first cleavage plane.
90
60
30
0
LD oils (N=43)
GD oils (N=43)
Figure 4. Polar graph showing departure in degrees of arc of lines
defined by oil drops injected at both ends of the lesser diameter (LD)
versus greater diameter (GD) of the zona pellucida (ZP) at the first
cleavage plane in 2-cell conceptuses from the equator of blastocysts
(0°). Note the conspicuously wider scatter for the GD than the LD.
35/43 (81%) blastocysts compared with only 22/43 (51%) for
the GD line. They, therefore, endorse an earlier finding that the
LD of the ZP at the first cleavage plane maps more consistently
equatorially on the blastocyst than the GD (Gardner, 2001a).
Hence, marking the LD of the ZP at the first cleavage plane
was adopted in further experiments to investigate whether the
ZP has a role in specifying the Em–Ab axis of the blastocyst.
Axial distribution of centres of patches produced by random
DiI-labelling
The critical experiments entail labelling a specific outer blastomere of advanced morulae with DiI and ascertaining the
axial location of the visual centres of the resulting patches of
fluorescence within the trophectoderm following blastulation.
Their interpretation obviously depends on how the distribution
of such centres compares with that expected if the choice of
outer blastomere for labelling were random. However, the centres of patches obtained by random labelling cannot be assumed
to map equally frequently within the Em polar, Eq and Ab polar
thirds of the Em–Ab axis because of complexities in the growth
of the trophectoderm during blastocyst expansion. Thus, during
the initial phase of blastulation, trophectoderm cells surrounding
the enlarging blastocoele become stretched so that <50% of
outer blastomeres in the late morula may contribute to the mural
as opposed to polar region in early blastocysts. With further
blastocyst expansion, these mural founder cells are evidently
consigned to the Ab polar trophectoderm where their proliferation declines relative to that in more proximal mural and polar
regions (Copp, 1978; Gardner and Davies, 2002). Finally, sustained mitotic activity in the polar trophectoderm produces a net
flow of cells into the proximal mural region (Copp, 1978; Cruz
and Pedersen, 1985) which is polarized rather than unrestricted
spatially (Gardner, 2000; Gardner and Davies, 2002).
It was therefore necessary to determine the distribution that
random labelling produces. Hence, advanced morulae cultured
in vitro from the 2-cell stage which, according to careful
inspection at ×500 using DIC optics, showed no sign of incipient cavitation were allowed to settle on the floor of individual,
strictly circular, hanging drops in a manipulation chamber. The
injection pipette was introduced into each drop and the outer
blastomere closest to its tip labelled with DiI. Thereafter, the
morulae were divested of the ZP with AT before being returned
to culture for development to the blastocyst stage. The distribution of the patch centres along the Em–Ab axis was clearly not
uniform (Figure 5, Table I), with the highest proportion occurring in the Eq and the lowest in the Ab one-third. To address the
possibility that the results might reflect failure to achieve a truly
random choice of blastomere for labelling with the ZP on, the
experiment was repeated on further advanced morulae that had
been completely denuded with AT before culture from the
2-cell stage. This yielded a distribution of centres that was so
similar to the one obtained with the ZP on (Figure 5 and Table I)
as to justify pooling of the two sets of control data to obtain the
proportions for calculating the numbers of centres expected to
map to the different thirds of the axis following labelling of a
specific outer blastomere. These proportions were 0.32, 0.43
and 0.25 for the Em, Eq and Ab thirds, respectively.
Pre-blastulation DiI-labelling of blastomere under LD oil
and ZP removal
In the first experiment, 2-cell conceptuses whose ZP was unequivocally oval at the first cleavage plane had a single large oil
drop injected at one end of its LD. The conceptuses were then
cultured to an advanced morula stage when they were returned
individually to hanging drops in a manipulation chamber
where they were inspected closely for any sign of intercellular
fluid accumulation. Those that had clearly not embarked on
blastulation had the outer blastomere immediately underlying
the oil drop in the ZP labelled with DiI. The ZP was then
removed with AT before the morulae were re-incubated for
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R.L.Gardner
Figure 5. Histograms showing the partitioning between the embryonic (Em), equatorial (Eq) and abembryonic (Ab) thirds of the Em–Ab axis of
blastocysts of centres of patches of DiI fluorescence in the trophectoderm produced by labelling an outer blastomere in advanced morulae (A–F).
Random outer blastomere labelling before removing the zona pellucida (ZP) (A) and following its removal at the 2-cell stage (B). (C) Combined
control data from (A) and (B). (D) Labelling of the outer blastomere underlying a lesser diameter (LD) oil in the ZP which was removed immediately thereafter. (E) Labelling of the outer blastomere underlying an LD oil in the ZP which had been softened with proteinase (PK) at the 2-cell stage.
(F) Labelling of outer blastomere underlying an intact second polar body (second PB) following removal of the ZP at the 2-cell stage.
recording the distribution of labelled cells following blastulation. The relative axial distribution in blastocysts of the centres
of the patches of DiI label was found to differ significantly
from that expected on the basis of the random controls (see
Figure 5D versus A–C and footnotes to Table I).
‘Softening’ the ZP at the 2-cell stage
It is evident from the foregoing experiment that, contrary
to the assertion of Motosugi et al. (2005), imposition of
802
mechanical constraint on the conceptus by the ZP during
blastulation fails to account for the non-random axial distribution in blastocysts of marker oil drops injected into the ZP
at the 2-cell stage (Gardner, 2001a). Nevertheless, it is conceivable that the ZP might play a critical role in enduringly
shaping the conceptus earlier in preimplantation development. This possibility was investigated by carrying out further double-marking experiments with the ZP ‘softened’ at
the 2-cell stage so as to prevent it exercising effective
mechanical constraint during subsequent preimplantation
Polarity specified before blastocyst stage
Table I. Summary of distribution of axial centres of DiI patches in the trophectoderm following labelling of an outer blastomere of advanced morulae
Treatment of conceptuses
Number scored
Embryonic 1/3
Equatorial 1/3
Abembryonic 1/3
Control 1: DiI-labelling of random outer blastomere followed by removal of ZP
Control 2: DiI-labelling of random outer blastomere after removal of the ZP at the 2-cell stage
Controls 1 and 2 combined
DiI-labelling of outer blastomere under LD oil injected into ZP at 2-cell stage
DiI-labelling of outer blastomere under LD oil injected into ZP which then softened at 2-cell
stage
DiI-labelling of outer blastomere under intact PB of conceptuses denuded at 2-cell stage
95
108
203
100
57
28
37
65
26
13
41
47
88
58
36
26§
24§
50
16*
8†
55
13
36
6‡
LD, lesser diameter; PB, polar body; ZP, zona pellucida.
Differs non-significantly: c2 for 2 degrees of freedom equal to 0.91: P > 0.5.
*Differs significantly from combined controls: c2 for 2 degrees of freedom equal to 9.57: P < 0.01.
†
Differs significantly from combined controls: c2 for 2 degrees of freedom equal to 9.54: P < 0.01.
‡
Differs significantly from combined controls: c2 for 2 degrees of freedom equal to 12.17: P < 0.005.
§
development. Various treatments including limited exposure
to AT proved unsatisfactory, because they caused thinning of
the ZP that was accompanied by conspicuous swelling of the
perivitelline space. However, culture of conceptuses in
mKSOM containing low concentrations of pronase or PK
(Lee et al., 1997) was found to increase the deformability of
the ZP without obviously reducing its thickness or enlarging
the perivitelline space. Following various trials, a 1.75-h
incubation of advanced 2-cell conceptuses in 10 μg/ml PK in
mKSOM was adopted. This produced obvious softening of
the ZP as judged by its increased deformability relative to
untreated specimens (Figure 6A). That this was sufficient for
the shape of the ZP to be altered by the enclosed conceptus
was then tested as follows. An oil drop was injected into the
ZP over the outer extremity of both blastomeres of a series of
72 2-cell conceptuses which were then photographed so as to
enable the distance between the centres of the two oil drops to
be measured with Image J software. Thereafter, the conceptuses were incubated for 1.75 h in mKSOM that either
contained or lacked PK before being transferred to pre-equilibrated calcium-free OC medium plus EGTA for a further 15
min to encourage rounding of their blastomeres. Each conceptus was then photographed once again for re-measuring
the distance between the two oil drops in the ZP (Figure 6B–
E). The mean initial and final distance in arbitrary units was
854.7 ± 5.98 versus 912.6 ± 5.98 for the 36 specimens in the
PK-treated series, representing an increase of 7% (P < 0.001),
and 863.7 ± 5.43 versus 876.6±5.56 for the 36 non-treated,
with an increase of ∼1% (P > 0.05). That treated ZPs did not
re-harden during the subsequent culture was evident from the
fact that surviving second PBs remained conspicuously
rounded even in blastocysts that had become well expanded.
Normally, they are invariably so flattened between the trophectoderm and ZP by this stage as to be hard to discern without
deflating the blastocoele. As shown in Figure 6F and G, the
PBs of well-expanded blastocysts developed from PK-treated
2-cell conceptuses retained their shape through locally
deforming the ZP rather than the trophectoderm. Moreover,
the overall shape of such ZPs tended to be less regular, and
the outer surface of trophectoderm cells was more rounded
than in untreated blastocysts.
To ascertain whether 1.75-h exposure to 10 μg/ml PK at
the 2-cell stage impaired development, a series of treated
conceptuses and untreated controls were transferred to opposite uterine horns of day 3 post-coitum pseudopregnant females
following culture in protease-free medium for a further 3 days.
The rates of implantation and post-implantation development
recorded 1 week later, which were similar for both treated and
untreated, are summarized in Table II.
Following exposure to PK, further 2-cell conceptuses with
an oil drop in the ZP to mark its LD at the first cleavage plane
were cultured to an advanced morula stage for DiI-labelling the
outer blastomere beneath the oil. After labelling, the morulae
were returned to culture without being exposed to AT, so that
any cases of marked subsequent rotation in the ZP could be
detected on examination at the blastocyst stage. Fifty-seven of
sixty-six scorable specimens (86%) showed correspondence of
the patch of fluorescence with the oil drop in the ZP and were
therefore photographed for scoring. The distribution of the centres of the patches along the Em–Ab axis again differed significantly from random being, as in the series with the ZP
untreated, relatively enriched equatorially and depleted in both
polar regions (Tables I and Figure 5E versus A–C).
Complete removal of the ZP at the 2-cell stage
Discounting the possibility that the ZP might still influence the
shape of the enclosed conceptus despite being softened
requires undertaking double-labelling experiments on specimens that have been denuded completely at the 2-cell stage.
For this, an alternative to oil drops in the ZP was obviously
needed as a marker for the plane of first cleavage. The second
PB seemed the most credible alternative, as it is almost invariably aligned with the cleavage plane at the 2-cell stage (Howlett
and Bolton, 1985), remains tethered to the conceptus and maps
preferentially to the Eq region of the blastocyst (Gardner,
1997). Two-cell conceptuses were therefore denuded carefully
with AT so as to minimize the risk of detaching the second PB
and inspected following culture to an advance morula stage.
Those with the second PB still intact had the blastomere immediately underlying it labelled with DiI before being re-incubated for scoring at the blastocyst stage. In accordance with the
earlier results, the centres of the patches produced by labelling
the blastomere beneath the second PB with DiI also showed
a significant Eq bias compared with that expected from the
controls (Tables I and Figure 5F versus A–C). Moreover, in all
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R.L.Gardner
17/55 cases where the second PB was still intact at scoring it
lay over the DiI-positive patch in the trophectoderm, clearly
showing that it had not suffered obvious relocation between the
advanced morula and blastocyst stages.
Discussion
Figure 6. Conceptuses with the zona pellucida (ZP) softened by
exposure to proteinase K (PK) (A–G). (A) A PK-exposed 2-cell conceptus (left) pushed against an untreated one (right) to show the greater
deformability of the treated ZP. (B–C) A PK-treated 2-cell conceptus
before (B) and after (C) incubation in calcium-free mKSOM with
EGTA to encourage the blastomeres to round up. Note the obvious
increase in distance between the pair of oil drops in (C) relative to
(B). (D–E) A second PK-treated 2-cell before (D) and after (E) incubation in calcium-free mKSOM with EGTA. In this case, some rotation of the conceptus in the ZP occurred between the photographs,
possibly because the greatest diameter of the ZP was initially more
nearly parallel than orthogonal to the first cleavage plane. (F–G).
Examples of well-expanded blastocysts developed from PK-treated
2-cell conceptuses whose second polar body (PB) is unusually prominent, causing local outward bulging of the ZP.
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The purpose of this study was to ascertain whether, as claimed
by Motosugi et al. (2005), the non-random mapping of the
2-cell conceptus on the blastocyst inferred from marking
experiments using oil drops in the ZP (Gardner, 2001a) is dictated by the shape of this glycoprotein coat. Thus, these workers
argue that the ZP is sufficiently resistant to deformation that
when it is obviously ellipsoidal rather than spherical it forces
the Em–Ab axis of the blastocyst to align with its greatest
diameter, either during blastocoele formation or thereafter. The
ZP is said to have its greatest diameter orthogonal to the plane
of first cleavage at the 2-cell stage and also to permit free rotation of the conceptuses during subsequent development.
Accordingly, the impression that the plane of first cleavage
maps to the equator of the blastocyst is held simply to be an
artefact of imposition of mechanical constraint by the ZP
(Motosugi et al., 2005). It is important to note that the ZP is
regarded as ellipsoidal rather than approximately spherical in
50–60% of conceptuses, so that the first cleavage plane should
be oriented randomly with respect to the blastocyst’s Em–Ab
axis in the remaining 40–50%. If these claims are indeed correct, the clear expectation from the double-labelling experiments
reported here is that the region of the conceptus underlying the
LD oil drop in the ZP at the advanced morula stage should bear
no fixed relationship to the Em–Ab axis, especially if the ZP is
removed before blastulation. This was not found to be the case.
The axial distribution of the centres of patches within the trophectoderm marked by labelling the blastomere underlying such
an oil drop was significantly non-random, with more centres
than expected equatorially and less that expected in the polar
regions. That this was not an artefact of DiI-labelling was
evident from the fact that it was not seen when an outer blastomere was chosen at random for labelling.
What does this finding signify? The oil drop under which the
blastomere was labelled marked one end of the ZP’s shortest
diameter at the plane of first cleavage. If, as Motosugi et al.
(2005) assert, the conceptus subsequently rotates freely in its
ZP, the region underlying the oil drop in the late morula should
seldom correspond to that underlying it at the 2-cell stage.
Moreover, in the absence of the ZP during blastulation, clonal
descendants of this blastomere should certainly not be
expected to show a preferential localization on the Em–Ab axis
of the blastocyst. Hence, the fact that they do argues against a
role for the ZP in specifying the orientation of the Em–Ab axis
of the blastocyst and clearly shows that the presence of this
coat during blastulation cannot account for the relationships
between the structure of the blastocyst and 2-cell inferred from
earlier oil drop marking experiments (Gardner, 2001a).
Nevertheless, providing it is not free to rotate, it is conceivable that the conceptus might be shaped enduringly by the ZP so
as to influence where the blastocoele eventually forms at an
earlier stage in preimplantation development. A further series
Polarity specified before blastocyst stage
Table II. Post-implantation development of proteinase (PK)-treated versus untreated control conceptuses
Treatment at 2-cell stage before culture to advanced
morula/blastocyst
Number transferreda
Number implanted
Number normal
Number retarded/abnormal
PK treated
Untreated
30
29
22
23b
17
19
1
3
a
Transferred in groups of 5–6 to opposite uterine horns of 5 day 3 pseudopregnant mice which became pregnant.
One decidua was empty.
b
of double-labelling experiments as therefore undertaken on
conceptuses whose ZP was softened enzymatically to an extent
that enabled them to affect its shape without impairing their
post-implantation development. The results showed that obvious rotation in the ZP between morula and blastocyst stages
occurred only occasionally and, more importantly, that the
axial distribution of centres of patches formed by the blastomere under the LD oil was also significantly non-random.
Although these findings make a role for the ZP during cleavage
in specifying the Em–Ab axis very unlikely, complete removal
of the ZP at the 2-cell stage was required to place this beyond
reasonable doubt. In the absence of the ZP, the blastomere
underlying an intact second PB in morulae was labelled with
DiI. Additional random blastomere injections were undertaken
to control for the modified conditions required for manipulating denuded morulae. As with labelling the blastomere under
the LD oil drop in the ZP, centres of the experimental fluorescent patches mapped significantly non-randomly with respect
to the Em–Ab axis relative to controls. This is particularly
interesting because the significant Eq bias in location of surviving second PBs at the blastocyst stage reported in an earlier
study (Gardner, 1997) is one of several findings which the
mechanical constraint hypothesis fails to explain. While
Hiiragi and colleagues make repeated reference to unpublished
observations purporting to show that movement of the second PB
is not necessarily constrained by its tether (Hiiragi and Solter,
2004, 2006; Motosugi et al., 2005), they have yet to provide
data to support this unlikely possibility. Confusion of the second
PB with the first PB seems an obvious source of error because,
judging from its lack of a tether and ease of relocation (Miao
et al., 2004; R. L. Gardner, unpublished observations), the
latter is attached to the conceptus much more tenuously than the
former. Moreover, the first PB not uncommonly survives intact
to the blastocyst stage and is not always distinguishable from
the second without recourse to nuclear staining (Gardner, 1997).
How accurately does the first cleavage plane map on the blastocyst? Despite showing significant Eq clustering, the centres of
the patches of DiI-labelling within the trophectoderm were distributed rather broadly along the Em–Ab axis of blastocysts in
all three series of experiments. This is not surprising in view of
unavoidable limitations in the resolution achievable by marking
individual outer blastomeres in advanced morulae. The blastomere for DiI-labelling was seldom centred under the oil drop
in the ZP or an intact second PB and could be sufficiently offset
as to make it difficult to decide whether it rather than an immediate neighbour should be labelled. Moreover, depending on
their orientation, subsequent divisions of both the labelled cell and
its neighbours may further increase the displacement of the centre of patches with respect to the Em–Ab axis of the blastocyst.
The patches may also suffer relocation in more advanced blastocysts because of the polar to mural flow of cells within the trophectoderm (Gardner, 2000; Gardner and Davies, 2002).
Although this study is therefore uninformative regarding the
variability with which the first cleavage plane maps on the
blastocyst, it does lend further credence to the interpretation of
earlier experiments in which the much better resolution
afforded by oil drops in the ZP was exploited for this purpose.
Thus, the findings reported here show that mapping of the
three oil drops marking the LD of the ZP at the cleavage plane
within the Eq one-third of the Em–Ab axis in 100/119 blastocysts (Gardner, 2001a) cannot depend on the presence of the
ZP. They also make significant rotation of the conceptus with
respect to this point in the ZP extremely unlikely, because it
would require that whatever part of the conceptus came to lie
under the LD oil drops before blastulation was thereby fated to
form part of the Eq region of the blastocyst. Rather, the results
strongly support the view that the plane of first cleavage is typically orthogonal to the Em–Ab axis of the blastocyst in a line
corresponding with the LD of the ZP at this plane.
Conflicting conclusions have been drawn using the boundary between clonal descendants of sister 2-cell stage blastomeres rather than oil drops in the ZP to map the first cleavage
plane on the blastocyst. Thus, although some claim that the
boundary tends towards orthogonal to the Em–Ab axis of the
blastocyst (Piotrowska et al., 2001:; Fujimori et al., 2003),
others have been unable to discern such a relationship (Alarcon
and Marikawa, 2003; Chroscicka et al., 2004; Motosugi et al.,
2005). As discussed elsewhere, given the extent to which this
boundary will vary according to the detailed pattern of cleavage and degree of mutual incursion of clones, this is hardly surprising (Gardner, 2005). An additional consideration emerging
from this study is that the relationship of the first cleavage
plane to the Em–Ab axis is likely to vary according to the orientation in which the blastocyst is viewed. Because findings
are also likely to vary according to strain, PO closed-bred mice
were chosen for this study as being more representative of the
species than inbreds or various hybrids between them.
The early mouse blastocyst is typically bilaterally rather
than radially symmetrical, having an oval rather than a circular
profile in polar view, with its LD corresponding with that of
the ZP (Gardner, 1997). Recently, the majority of advanced PO
strain zygotes have also been found to be overtly bilaterally
symmetrical with their bilateral plane approximately parallel to
the zygote’s animal–vegetal axis (Gardner and Davies, 2006). Not
only is the first cleavage plane very consistently orthogonal to
the zygote’s bilateral plane, but the bilateral plane of the blastocyst is aligned with it. Consequently, the LD of the ZP at the
first cleavage plane is usually orthogonal to the bilateral planes
805
R.L.Gardner
References
A
B
Figure 7. Two diagrammatic views of a bilaterally symmetrical
advanced mouse zygote. (A) Animal polar view with the bilateral
plane indicated by a thick line, and the future first cleavage plane
indicated by the darker oval shape with a dashed outline. The arrows
denote that although the first cleavage plane is consistently orthogonal to the bilateral plane, it is variable in orientation with respect to
the zygote’s animal-vegetal axis whose animal pole is defined by the
position of the second polar body depicted in white. (B) View of the
same specimen with its bilateral plane horizontal and hence the offaxis first cleavage plane vertical, as shown by its dashed outline.
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The challenge ahead is to elucidate the nature of prepatterning
and determine to what extent it may depend on processes
occurring before fertilization as opposed to thereafter.
Acknowledgements
I thank Tim Davies and Andrea Kastner for help and both the
March of Dimes Birth Defects Foundation and the Royal Society
for support.
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Submitted on July 7, 2006; resubmitted on September 20, 2006; accepted on
September 29, 2006