Human Reproduction Vol.21, No.2 pp. 492–502, 2006
doi:10.1093/humrep/dei318
Advance Access publication October 6, 2005.
An investigation of the origin and significance of bilateral
symmetry of the pronuclear zygote in the mouse
R.L.Gardner1 and T.J.Davies
Mammalian Development Laboratory, University of Oxford, Department of Zoology, South Parks Road, Oxford OX1 3PS, UK
1
To whom correspondence should be addressed. E-mail: [email protected]
BACKGROUND: Preliminary observations revealed that advanced zygotes of the PO strain mouse are often bilaterally
symmetrical, and suggested that both the plane of first cleavage and features of the blastocyst bear a consistent
relationship to the zygote’s bilateral plane. METHODS: Spaced oil drops were injected into the zona pellucida to
delineate the bilateral plane in pronuclear zygotes, and a distinct cluster of drops then placed over the second polar
body. Such non-invasive marking was combined with gelation of the perivitelline space to prevent rotation of the
zygotes within the zona pellucida. RESULTS: Nearly two-thirds of advanced pronuclear stage zygotes were bilaterally symmetrical and, regardless of whether first cleavage was meridional, it was almost invariably orthogonal to the
bilateral plane. Moreover, both the axis of polarity and bilateral plane of the blastocyst bore a consistent relationship
to the zygote’s bilateral plane. Haploid parthenotes also exhibited bilateral symmetry, although in the absence of fertilization, first cleavage was less consistently orthogonal to the bilateral plane. CONCLUSIONS: Bilateral symmetry
may be an intrinsic property of the oocyte that is induced by its activation and, from the reproducible way it maps on
both the 2-cell conceptus and blastocyst, seems to play a role in early patterning.
Key words: bilateral symmetry/blastocyst/first cleavage plane/pre-patterning/zygote
Introduction
Recently, Hiiragi and Solter (2004) have argued that, contrary
to the findings from various other studies (e.g. Gardner, 1997,
2001; Piotrowska et al., 2001; Fujimori et al., 2003), specification of axes cannot normally occur during the zygotic stage of
development in the mouse. They base this conclusion on their
confirmation by time-lapse recording of an earlier finding that
first cleavage is often clearly non-meridional, with the second
polar body (2nd PB) moving to its plane during the course of
division (Gardner and Davies, 2003a). Since, by convention,
the second polar body defines the animal pole of the zygote
(Balinsky, 1970), its movement is said to rule out the existence
of a developmentally significant animal–vegetal (AV) axis
(Hiiragi and Solter, 2004).
Hiiragi and Solter (2004) claim that the plane of first cleavage is invariably orthogonal (i.e. perpendicular) to the line in
which male and female pronuclei come together, and that the
latter is dictated by the relationship of the site of sperm entry
to that of production of the 2nd PB. It is, however, hard to
evaluate this claim because, in the absence of any data showing the precision with which the plane orthogonal to this line
could be determined, it is impossible to judge how closely
cleavage followed it. In our experience, it is not easy to decide
the orientation of a plane that is tangential to the point of
apposition of an adjacent pair of dissimilar-sized late pronuclei
whose boundaries are becoming ill-defined. Moreover, Hiiragi
and Solter (2004) make no mention of findings which suggest
that first cleavage is typically orthogonal to a plane of bilateral
symmetry that is discernible in the majority of advanced zygotes
(Gardner and Davies, 2003a). These findings clearly prompt the
question of how pronuclear migration relates to bilateral symmetry of the zygote. Does the plane in which the pronuclei lie specify the bilateral plane of the zygote, or vice versa? If the former
is the case and pronuclei migrate directly from their site of
formation to the centre of the zygote, as Hiiragi and Solter
(2004) assume, the bilateral plane could be defined by the relationship of the site of sperm entry to that of production of the
2nd PB. If the latter, bilateral symmetry would appear to be a
manifestation of intrinsic organization of the oocyte that dictates
the plane in which the pronuclei eventually come to lie.
The main purpose of the present investigation was to confirm and extend our studies on the relationship of the plane of
first cleavage to that of bilateral symmetry of the zygote, and to
ascertain how the pronuclei relate to the bilateral plane.
Materials and methods
All oocytes and most zygotes were obtained from the PO albino noninbred strain of mice that were kept under a 24 h lighting regime with
the dark period from 19:00–05:00h or 11:00–21:00h. The remaining
zygotes were from MF1 non-inbred albino mice whose daily dark
period was from 13:00–23:00h. Except where stated otherwise, all
492 © The Author 2005. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
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Bilateral symmetry of the mouse zygote
observations relate to the PO strain. In view of continuing concern
about adverse effects on development of its hormonal induction
(Ertzeid and Storeng, 2001), ovulation was allowed to occur naturally
throughout this study. For obtaining zygotes and oocytes, females
were placed with fertile and vasectomized males, respectively, before
the onset of the dark period and checked for vaginal plugs thereafter.
Unless indicated otherwise, media and conditions used for recovery, storage, manipulation and culture of oocytes, zygotes and cleavage stages were as reported earlier (Gardner, 2001; Davies and
Gardner, 2002). Medium or low viscosity sodium alginate (Sigma,
UK) was prepared at various concentrations in potassium simplex
optimized medium (KSOM) medium lacking calcium. For gelling the
perivitelline space (PVS), zygotes were incubated for up to 1.5 h in
this alginate-containing medium, then rinsed for 1–2 min in KSOM–
HEPES before being transferred for 15–20 min at room temperature to
an aliquot of the same medium supplemented with 0.3% CaCl2·2H2O.
Pronase (Calbiochem) was prepared as a 0.5% solution in Dulbecco
A phosphate-buffered saline, sterilized by filtration and frozen in 200
μl aliquots which were centrifuged briefly at 11 000 ×g immediately
before use to sediment particulate material.
Surface features of zygotes were marked strictly non-invasively by
injecting drops of soya oil into the zona pellucida (ZP) (Gardner,
2001). The bilateral plane was outlined with either a complete or partial ring of well-spaced drops, and the site of the 2nd PB with a focal
cluster of three or so additional drops. Zygotes were then cultured for
analysis at either or both the 2-cell and blastocyst stages. Measurements were made on a monitor coupled to a low-light camera mounted
on the phototube of the micromanipulation microscope, or from prints
of photographs taken with a similarly mounted 35 mm camera.
Oocytes in cumulus were activated artificially by exposing them to
strontium, as described by Fraser (1987), and production of a 2nd PB
was taken as evidence of successful induction of haploid parthenogenetic development.
For ascertaining their stage of mitosis, advanced zygotes whose
pronuclei were no longer visible were incubated in standard culture
medium containing Hoechst 33342 at 5 μg/ml for 5–10 min before
being examined by fluorescence microscopy.
Results
Incidence and patterns of bilateral symmetry among zygotes
The majority of advanced pronuclear stage PO zygotes are
oval rather than circular in outline when viewed with the 2nd
PB uppermost, and can thus be assigned two planes of bilateral symmetry that correspond with their greater and lesser
diameter in polar view. The plane corresponding with their
greater diameter is referred to hereafter as the bilateral plane.
The profile of most such zygotes is roughly circular when
they are viewed with their bilateral plane horizontal rather
than vertical so that they are approximately oblate spheroids
in overall shape (see Figure 1A and D and Figure 2A–D). A
substantial minority exhibit a markedly oval profile in this
view through being obviously elongated parallel to their
bilateral plane, most commonly obliquely to their AV axis
(Figure 1F). They therefore resemble oblate spheroids that
have been distorted by restricted directional stretching
(Figure 1E–G and Figure 2F and G). The proportion of
advanced pronuclear stage PO zygotes that were already
bilaterally symmetrical on recovery from the oviduct varied
considerably between donors, with the highest being 10/11.
The mean proportion was 32% (176/556) which, depending
Figure 1. (A, B) When rotated about its lesser diameter (dashed line
in A), an oval or ellipse defines the shape of an oblate spheroid and,
when rotated about its greater diameter (dashed line in B), a prolate
spheroid. Consequently, oblate and prolate spheroids differ in having
a circular rather than oval profile when viewed, respectively, along
their lesser and greater diameters. Bilaterally symmetrical zygotes are
typically oval when their bilateral plane is vertical (C), but rather variable in shape when it is horizontal (D–G). Many have a roughly circular profile in side view (D) and thus approximate to oblate spheroids
in overall shape. The remainder are more or less oval in lateral view
(E–G), but differ in orientation of their greater diameter relative to the
animal–vegetal (AV) axis. The ratio of the lesser to greater diameter
in polar and side view of the relatively uncommon C–G category
very seldom corresponds sufficiently closely for this category to
qualify as a prolate spheroid. Hence, all categories except C–D are
essentially oblate spheroids that are distorted by being elongated parallel (C–E), obliquely (C–F), or orthogonally (C–G) to the AV axis.
Among these, the most common is C–F (Figure 2F and G), and the
least common, C–G.
mainly on observations at the relatively low magnification
afforded by a dissecting microscope, is a minimal estimate.
Overall, the incidence of bilateral symmetry among advanced
pronuclear stage PO zygotes in culture was 63% (403/641). For
a more limited number of MF1 zygotes that were examined, the
incidence of bilateral symmetry (Figure 2E) was 62% (61/99).
The proportion of bilateral PO zygotes that were obviously
elongated rather than simply oblate was 30% (121/403), and
those whose bilateral plane was orthogonal or oblique to the AV
axis or which were obviously prolate amounted collectively
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R.L.Gardner and T.J.Davies
Figure 2. (A–B) A modestly oblate zygote in polar view (A) with its bilateral plane marked with an opposed pair of oil drops in the zona pellucida (ZP), and in lateral view (B) to show its profile is approximately circular with the bilateral plane horizontal. Though the pronuclei are
somewhat out of focus in (A), they are clearly not aligned with the bilateral plane. (C–D) A more conspicuously oblate zygote viewed from
the animal pole (C), and with its bilateral plane horizontal (D). (E) Polar view of a bilaterally symmetrical MF1 zygote. (F–G) Lateral views
of two PO zygotes which, according to the position of the second polar body and in contrast to those depicted in A–D, are elongated obliquely
to the animal–vegetal axis. The partial ring of spaced oil drops in the ZP delineates the bilateral plane. Note that the two pronuclei lie in the
bilateral plane in G but not in F. Despite their irregular shape, such elongated zygotes form 2-cell conceptuses that are indistinguishable from
those produced by regularly oblate specimens and develop similarly to the blastocyst stage. (H) An advanced pronuclear zygote that is not
perceptibly bilateral in polar view.
to 7% (30/403). Thus, a majority of pronuclear zygotes of PO
and MF1 mice clearly become bilaterally symmetrical, but it
remains uncertain whether all do so at some stage (Figure 2H).
Mapping of the bilateral plane of the zygote on the 2-cell
stage
A first step in trying to determine the significance of bilateral symmetry of the zygote was to see how it maps on subsequent stages of development, and this requires enduring
marking of the zygote’s bilateral plane. There is still no
convincing evidence that exogenous labels applied to the
surface of zygotes retain their position through first cleavage. Indeed, that so large an anchored structure as the 2nd
PB often moves a considerable distance towards the cleavage plane as the zygote divides (Gardner and Davies, 2003a;
Hiiragi and Solter, 2004) makes achieving reliable surface
marking a doubtful prospect. However, strictly non-invasive
marking with small drops of mineral oil injected into the ZP
enabled surface features of the 2-cell conceptus to be related
to the structure of the blastocyst in the mouse (Gardner,
2001). This was because the conceptus evidently undergoes
little if any rotation within its ZP during development in
vitro between these stages.
The bilateral plane was therefore ringed with small oil
drops injected into the ZP of a series of zygotes that were
clearly bilaterally symmetrical in polar view. An additional
cluster of three or so drops was placed over the 2nd PB to
check whether the conceptus remained in register with its ZP
through first cleavage (see Figure 2F and G). In the 22 cases
where the 2nd PB was still under its marker oil drops at the 2-cell
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stage, cleavage showed an intriguingly non-random relationship to the ring of oil drops used to delineate the zygote’s
bilateral plane, being almost invariably closer to orthogonal
than parallel to the latter (Figure 3A). A trivial explanation
for this finding is that temporary adoption of an oblate or
elongated form by a zygote serves to mould its ZP in a corresponding shape. Hence, when the zygote lengthens during
anaphase it tends to rotate until its long axis accords with the
greater diameter of the ZP, and thus with the latter’s ring of
oil drops. Providing cleavage was accurately meridional and
the zygote turned about its AV axis, such rotation could occur
without the PB obviously shifting position with respect to its
marker oil drops. That enduring moulding of the shape of the
ZP does occur was confirmed by making a modest slit in it
through which the zygote could be removed by suction via a
holding pipette. The greater diameter of such evacuated ZP
typically corresponded with the oil ring marking the zygote’s
bilateral plane (Figure 4A and B).
To address the possibility that the effect of bilateral symmetry on cleavage was mediated by the ZP, the foregoing marking
experiments were repeated using gelation of the PVS with alginate to prevent zygotes rotating within this mucoprotein coat.
Initially, incubation in mKSOM supplemented with 1.1%
sodium alginate of medium viscosity was used to obtain accumulation of the polysaccharide in the PVS. Later, low viscosity
sodium alginate was used at concentrations between 0.8 and
1.5%. Importantly, zygotes that were radially symmetrical
when the PVS was gelated were not thereby prevented from
becoming bilaterally symmetrical thereafter. However, presence of the gel did not ensure that the 2nd PB invariably
remained at its original location following first cleavage. Not
Bilateral symmetry of the mouse zygote
Figure 3. (A–D) Histograms showing the angular relationship
between the first cleavage plane and a partial ring of oil drops in the
zona pellucida (ZP) defining the bilateral plane of advanced zygotes.
Angles at 10° intervals (abscissa) versus numbers of zygotes (ordinate). (A) Relationship of the first cleavage plane to the oil ring in
cases where the second polar body was still under its cluster of marker
oil drops following marking of the bilateral plane of zygotes that were
free to rotate within their ZP. (B) Ringing of the bilateral plane in PO
zygotes whose perivitelline space (PVS) was gelated prior to first
cleavage. (C) Ringing of the bilateral plane in MF1 zygotes whose
PVS was gelated prior to first cleavage. (D) Ringing of the bilateral
plane in PO zygotes whose ZP was thinned with pronase. The numbers in brackets are specimens in which the PB was closer to the
cleavage plane than its marker oil drops but nonetheless remained
aligned with the ring.
infrequently, this body lay closer to the cleavage plane than its
marker oils but was nevertheless still well aligned with the ring
of drops used to demarcate the zygote’s bilateral plane. This
was assumed to be due to the well-authenticated shift of the
2nd PB towards the plane of cytokinesis that occurs where first
cleavage is obviously non-meridional (Gardner and Davies,
2003a; Hiiragi and Solter, 2004) rather than to the conceptus
rotating within its ZP. That its marker oils were very seldom
closer to the cleavage plane than the 2nd PB itself accords with
this assumption, as does the finding that a disparity in location
between the 2nd PB and its oil drops was much less common
when the latter were near the cleavage plane than well away
from it. Moreover, an obvious local outward bulging of cytoplasm beneath the marker oils was sometimes evident, consistent with spreading of the adjacent blastomere to fill the gel-free
space from which the 2nd PB had been displaced. Nonetheless,
rotation was evident in the minority of cases where the 2nd PB
was out of register with its marker oils and the bilateral ring of
drops both parallel as well as orthogonally to the cleavage
plane. The findings for cases where rotation of the conceptus
within its ZP could confidently be discounted are summarized
in Figure 3B. They reveal a striking tendency for the cleavage
plane to be orthogonal to that of bilateral symmetry of the
zygote (Figure 4C–F), notwithstanding inclusion of a substantial number of very modestly bilateral specimens. Particularly
notable are numerous cases where the 2nd PB remained under
its marker oils even though first cleavage was conspicuously
non-meridional (Figure 4G–H). Here, the possibility that conceptuses had rotated about the AV axis seems very remote. It is
also noteworthy that the relationship of cleavage to the bilateral
plane was similar regardless of the length of the interval
between oil drop ringing and mitosis. The relationship of first
cleavage to the ringed bilateral plane in a series of MF1 zygotes
in which rotation within the ZP could be discounted is shown in
Figure 3C. Before the notion that the cleavage plane is directly
related to the plane of bilateral symmetry of the zygote can be
entertained seriously, two further alternative possibilities need
to be considered. The first also presupposes that bilateral symmetry of the zygote serves merely to shape the ZP. However,
rather than rotating within its ZP during mitosis, the zygote may
probe the space around it and orient the mitotic spindle in the
direction in which its elongation is least restricted. This possibility was investigated by ‘softening’ the ZP so that its shape was
dictated by that of the zygote rather than vice versa. For this,
zygotes were incubated for 4.0 min at room temperature in
0.5% pronase in PBS. They were then transferred via a rinse in
KSH to calcium-free KSOM containing 0.8% low viscosity
sodium alginate. Incubation in this medium for ≥50 min encouraged partial collapse of the thinned ZP onto the surface of the
vitellus. Finally, the zygotes were transferred via a KSH rinse
without exposure to elevated calcium, to manipulation chamber
drops for marking of their bilateral plane and 2nd PB with oil
drops in the ZP. Weakening of the ZP was obvious from the difference in the shape of enzyme-treated and untreated specimens
following first cleavage (Figure 4I and J versus C–H).
The results, which are presented in Figure 3D, show that the
cleavage plane was approximately orthogonal to the bilateral
plane in the great majority of cases, although obvious exceptions
were more common than when the PVS was gelated with alginate.
This may reflect differences in the degree to which the ZP was
weakened which was apparent from its variable appearance. Interestingly, pronase treatment reduced the frequency with which the
2nd PB shifted towards the cleavage plane from under its marker
oil drops in the ZP. It also resulted in focal accumulation of a conspicuous mass of granular material at this plane (see Figure 4I and
J) whose nature is considered further in the Discussion.
Further evidence against a role for the ZP in orienting first
cleavage came from observations on occasional zygotes with
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Figure 4. (A–B) Evacuated zonae pellucidae (ZP) showing continued correspondence of their greater diameter with the ring of oil drops
used to delineate the bilateral plane of the zygote before it was removed. (C–D) 2-Cell conceptus with the plane of first cleavage orthogonal
to the ring of oil drops used to demarcate the bilateral plane at the zygote stage in polar view (C), and in side view (D) to show the polar
body (PB) is still under its marker oil drops. (E–F) Another 2-cell conceptus showing orthogonal relationship of the cleavage plane to the
ring of oils defining the zygote’s bilateral plane in polar view (E) and 2nd PB under its marker oil drops in side view (F). (G–H) Two specimens showing well ‘off-axis’ first cleavage with the PB still under its marker oil drops. (I, J) Examples of 2-cell conceptuses whose ZP
was thinned by pronase at an advanced zygote stage. Note how the shape of the closely investing ZP conforms to that of the conceptus compared with untreated specimens (C–H). Also note the accumulation of granular material at cleavage plane (arrowed). (K, L) Differential
interference contrast (K) and fluorescent Hoechst 33342 (L) images of an overtly bilateral zygote that regained a strictly circular profile
before the condensing maternal and paternal sets of chromosomes have actually come together.
the PVS gelated whose bilateral plane did not accord with greater
diameter of the ZP. Here, the cleavage plane was consistently
closer to orthogonal to the zygote’s bilateral plane than the ZP’s.
Moreover, unequivocally bilateral zygotes whose ZP was not
perceptibly oval were encountered regularly in the course of the
PVS gelation experiments, and these also typically exhibited an
orthogonal relationship of the cleavage plane to the bilateral ring.
Especially since ringing the ZP with oil drops depends on
holding the zygote by suction at one or more sites centred on
the plane in which the ring of oil drops is made, a second further possibility is that the observed relationship is simply an
artifact of micromanipulation. This was addressed by making
an oil ring in the ZP parallel to the AV axis in zygotes with the
PVS gelated that were not perceptibly oval in polar view (see
Figure 2H). The relationship of the cleavage plane to the oil
ring was strikingly different from that obtained following ringing of the bilateral plane (cf. Figures 3B and 5A).
Is persistence of bilateral symmetry required to orient first
cleavage?
Collectively, the above findings argue that the observed relationship of the plane of first cleavage to that of bilateral symmetry of
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the zygote is neither a consequence of moulding of the shape of
the ZP nor an artefact of micromanipulation. The orientation of
first cleavage seems therefore to be specified in relation to bilateral
symmetry that the zygote acquires several hours earlier during its
pronuclear stage. In considering how specification might be mediated, one obvious possibility is that bilateral symmetry persists
through to mitosis and thus serves to orient the long axis of mitotic
spindle in accordance with Hertwig’s rule (Wilson, 1928). Hence,
further zygotes with their bilateral plane ringed were observed at
intervals of 20–30 min in order to identify any that re-acquired
radial symmetry following pronuclear membrane breakdown. The
relationship of the plane of first cleavage to that of bilateral symmetry was recorded in these specimens and, to control for the possibility that the orientation of cytokinesis might be perturbed by
examining specimens repeatedly as they approached mitosis, also
in others that remained bilaterally symmetrical. Regardless of
whether the bilateral plane was ringed before or after gelation of
the PVS, some zygotes were no longer discernibly bilateral by the
time the pronuclei had disappeared (Figure 4K and L). Interestingly, the relationship of the plane of first cleavage to the oil ring
was not dissimilar in these specimens from those retaining bilateral symmetry (Figure 5B). Hence, once it has been established,
the bilateral condition can evidently persist in cryptic form.
Bilateral symmetry of the mouse zygote
Figure 5. (A) Histogram showing the angular relationship between the first cleavage plane and a partial ring of oil drops in the zona pellucida
(ZP) defining a random animal–vegetal plane in zygotes with the perivitelline space gelated which, like the specimen shown in Figure 2H, were
not perceptibly oval in polar view. (B) Angular relationship of the first cleavage plane to the bilateral plane in zygotes that fully rounded up prior
to mitosis (black columns) versus those that did not (hatched columns). Abscissae and ordinates are as in the legend to Figure 3.
Mapping of bilateral plane of zygote on the blastocyst
A series of advanced zygotes with the bilateral plane ringed
following gelation of the PVS were cultured to the blastocyst
stage, either continuously or with a brief interruption for
scoring at the 2-cell stage. The oil ring not only showed a
marked tendency to align with the blastocyst’s embryonic–
abembryonic (Em.Ab) axis (Figures 6A and 7A), but was,
moreover, approximately parallel to its greater diameter in all
nine blastocysts that were unequivocally bilaterally symmetrical (Figure 6B). The latter finding prompted extension of
these ringing experiments with the aim of increasing the proportion of blastocysts that were scored early enough for bilateral symmetry to be obvious (Gardner, 1997). In this further
series, measurements on the alignment of the bilateral plane
of the blastocyst with that of the zygote were made on
unequivocally bilateral specimens many of which had the 2nd
PB or its remnant still beneath its marker oils in the ZP. The
findings are summarized in Figure 7B. Although typically
well aligned, the two planes were not always accurately parallel to each other, a feature that is evident from the relationship of the Em.Ab axis of the blastocyst to the zygote’s
bilateral plane shown in Figure 7A. Possible explanations for
this are considered in the Discussion.
Relationship of bilateral symmetry to arrangement of
pronuclei
Hiiragi and Solter (2004) claim that the plane of first cleavage
is invariably orthogonal to the line of apposition of male and
female pronuclei once they have migrated to the centre of the
zygote. If this is indeed the case then, according to present
findings, this line must also correspond with the zygote’s
bilateral plane. This requires either that the bilateral plane is
established in conformity with the deployment of the pronuclei
or vice versa. Deciding between these two possibilities
depends on ascertaining how the pronuclei are aligned in relation to the bilateral plane when this plane is first established.
Coincidence of both from when bilateral symmetry is first evident would support dependence of the bilateral plane on pronuclear relations. In practice, whether or not the pronuclei were
apposed, the line joining their visual centres bore a highly variable relationship to the bilateral plane of the zygote (see Figure
2A, C and E, Figure 6C–F), even when they were becoming
obscured in anticipation of mitosis (Figure 7C). Finally, incidental observations on the relationship of male and female pronuclei to their sites of origin offered no support for the notion
that they migrate directly to the centre of the zygote.
Even if the site of sperm entry bears too variable a relationship
to the plane of first cleavage to play a role in orienting bilateral
symmetry (Davies and Gardner, 2002), it might nonetheless be
indispensable for its induction. This possibility was explored by
examining oocytes that had been activated to develop parthenogenetically through exposure to strontium whilst they were still
encased in cumulus (Fraser, 1987). Taking production of the
2nd PB as evidence of activation, the majority of the resulting
pronuclear stage parthenotes were found to be oval in polar
view, though generally less conspicuously so than zygotes
obtained by fertilization. These haploid parthenotes were cultured through first cleavage following gelation of the PVS and
oil drop marking of the bilateral plane plus site of the 2nd PB.
Although the relationship between cleavage and bilateral planes
varied more than following fertilization, the results clearly
show the same trend (Figure 7D). In contrast, even when incubated for up to 48 h, very few of several hundred oocytes that
had neither been exposed to strontium nor had undergone spontaneous activation were discernibly oval in polar view.
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Figure 6. (A) Lateral view of blastocyst showing correspondence of its embryonic–abembryonic axis with the ring of oil drops used to define
the bilateral plane of the zygote. (B) Polar view of a bilaterally symmetrical blastocyst with the ring of oil drops defining the bilateral plane of the
zygote aligned with its greater diameter. (C–F) Polar view of zygotes whose pronuclei are clearly not aligned with the bilateral plane which is
indicated in the less obviously oval specimens by a pair of white lines (C) or opposed oil drops (F).
Discussion
Once their pronuclei are well developed, the majority of
advanced pronuclear stage zygotes of both PO and MF1 mice
are unequivocally bilaterally rather than radially symmetrical.
Whilst most bilateral zygotes are approximately oblate (see
Figures 1 and 2), a substantial minority are obviously elongated in side view, usually obliquely to the AV axis. The
results of the present study fully support earlier preliminary
findings which suggested that first cleavage is typically orthogonal to the plane of bilateral symmetry of the pronuclear stage
zygote (Gardner and Davies, 2003a). They also show that this
relationship is neither mediated by the zygote imposing a corresponding change in shape on its investing ZP, nor attributable to manipulations required to mark the bilateral plane.
Confirmation has also been obtained that the blastocyst’s
Em.Ab axis tends to align with the zygote’s bilateral plane.
Most importantly, the present study also strongly endorses
earlier preliminary evidence that the bilateral plane of the blastocyst is consistently aligned with that of the zygote (Gardner
and Davies, 2003a). That these stages should be related with
respect to such a fundamental attribute makes it most unlikely
that reproducible mapping of features of the zygote and 2-cell
conceptus on the blastocyst can be attributed simply to the
operation of unspecified topological constraints during cleavage (Hiiragi and Solter, 2004; Rossant and Tam, 2004).
While the majority of advanced pronuclear stage zygotes
were unquestionably bilaterally rather than radially symmetrical, a variable minority were not. This may reflect asynchrony
in development or variability in the length of time for which
bilateral symmetry endures. A further possibility is that all
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zygotes acquire a bilateral internal organization that is not necessarily manifest as a change in their outward form. This could
account for why the orthogonal relationship of cleavage to the
bilateral plane was evident even in zygotes that ceased to be
overtly bilaterally symmetrical prior to cleavage (Figure 4).
That such zygotes were somewhat more variable in this respect
than those in which bilaterality was maintained may be
because their bilateral plane was typically less conspicuous,
and therefore harder to ring accurately.
The present findings challenge the conclusion of Hiiragi and
Solter (2004) that the plane of first cleavage depends solely on
the orientation in which the two pronuclei become apposed.
That the relationship of cleavage to the bilateral plane was similar regardless of how long before mitosis oil drop marking of
the ZP was done argues that bilateral symmetry is stable from
when it can first be discerned. Regardless of whether the pronuclei were apposed, the line joining their visual centres did
not show preferential alignment with the bilateral plane, as
would be expected if cleavage were invariably orthogonal to
this line (Hiiragi and Solter, 2004). This was true even after
pronuclear membrane breakdown had started (see Figure 7C).
Hence, if the relationship of cleavage to the line of pronuclear
apposition is as invariant as Hiiragi and Solter (2004) assert,
one must assume that the pronuclei come to lie in the bilateral
plane only shortly before mitosis. Consequently, contrary to a
further assertion by these authors, orientation of the plane of
first cleavage cannot be dictated by the spatial relationship of
the site of sperm entry to that of production of the 2nd PB.
Rather, its specification is evidently linked to bilateral symmetry of the zygote which, according to the findings of Hiiragi
Bilateral symmetry of the mouse zygote
Figure 7. (A, B) Angular relationship between the oil ring demarcating the bilateral plane of the zygote and the embryonic–abembryonic
axis (A) and bilateral plane (B) of the blastocyst. In (B), the black part
of the bars represents specimens whose 2nd PB remain under its
marker oil drops. (C) Angular relationship between pronuclear orientation and the bilateral plane in zygotes with pronuclei that were very
late (black), apposed (grey), and not apposed (white). (D) Angular
relationship between first cleavage and the bilateral plane in haploid
parthenotes. Abscissae and ordinates are as in the legend to Figure 3.
and Solter (2004), would require eventual alignment of the two
pronuclei within the bilateral plane. However, the plane of first
cleavage has been reported very recently to be typically parallel
rather than orthogonal to the line of apposition of the pronuclei
(Plusa et al., 2005). These findings, which directly contradict
those of Hiiragi and Solter (2004), would obviously require the
pronuclei to lie in a plane that was orthogonal to the zygote’s bilateral plane. Lack of a consistent alignment of pronuclei relative to
the plane of first cleavage has also been reported for human
zygotes (Gianaroli et al., 2003; Kattera and Chen, 2004), implying
that rotation of the mitotic spindle must occur following pronuclear membrane breakdown in many such zygotes (Scott, 2000).
While alignment of pronuclei with respect to the bilateral plane
might account for first cleavage being orthogonal to this plane, it
leaves open the question of what determines how cytokinesis is
oriented relative to the zygote’s AV axis. Although frequently
non-meridional, first cleavage is clearly not completely random
with respect to the AV axis (Gardner and Davies, 2003a; Gray
et al., 2004; Hiiragi and Solter, 2004). It is, nevertheless, often
sufficiently far ‘off-axis’ to make it unlikely that the mid-plane of
the first mitotic spindle is oriented by an entity close to the 2nd PB
such as its midbody (Plusa et al., 2002a). Hence, what dictates
which of the many possible planes orthogonal to the zygote’s
bilateral plane cleavage actually follows remains uncertain.
Since it is evidently instrumental in both specifying the orientation of first cleavage and patterning the blastocyst, it is of interest to know whether bilateral symmetry is an intrinsic property of
the oocyte or one that is imposed on it through events relating to
fertilization. Here, it is relevant to note claims based on surface
marking experiments that the site of sperm entry is more reliable
than the 2nd PB as a predictor of the plane of first cleavage (Piotrowska and Zernicka-Goetz, 2001; Plusa et al., 2002b). More
recently, the plane of first cleavage was reported to lie within 30°
of the centre of the fertilization cone in 19/31 [C57BL/
6×CBA]F2 zygotes whose development was recorded by timelapse (Gray et al., 2004). However, no consistent relationship
between cleavage plane and the site of sperm entry was found
using the anterior end of the endocytosed tail of the fertilizing
sperm as a marker (Davies and Gardner, 2002). Critiques of
these disparate findings are presented elsewhere (Gardner and
Davies, 2003b; Johnson, 2003; Zernicka-Goetz, 2003). According to the present findings, for the site of sperm entry to be
involved in specifying the plane of first cleavage it would have to
play a role in orienting as well as inducing bilateral symmetry,
and should thus be expected to bear a consistent relationship to
the bilateral plane. Gray et al. (2004) reported a tendency of the
fertilization cone to be orthogonal to the greater diameter of early
zygotes which were subtly oval in polar view. However, observations were limited to few specimens and the relevance of this
early shape change to the obvious bilateral symmetry of later PO
and MF1 zygotes has yet to be explored. No relationship
between the sperm entry site and bilateral plane was discernible
in late zygotes in which the anterior end of the tail of the fertilizing sperm, which has been validated as a marker of the sperm
entry site (Davies and Gardner, 2002), was still visible. Hence,
the role of the fertilizing sperm seems to be confined to inducing
rather than organizing bilateral symmetry, presumably as a facet
of its activating role. This accords with the finding that unfertilized oocytes routinely developed bilateral symmetry on culture
only following activation by exposure to strontium. However,
bilateral symmetry tended to be less conspicuous in haploid parthenotes than in zygotes produced by fertilization, and the relationship of cleavage to the bilateral plane was also more variable.
In a previous study, oil drops in the ZP were used as markers
for mapping features of the 2-cell conceptus on the blastocyst
stage. Drops placed over the outer extremity of one 2-cell blastomere typically mapped to either end of the Em.Ab axis of the
blastocyst, while those placed over the cleavage plane mapped
to its equator (Gardner, 2001). This is as expected if the bilateral plane of the blastocyst accords with that of the zygote and
first cleavage is usually orthogonal to the latter. Possible explanations for why use of lineage-labelling of one or both 2-cell
blastomeres to map the plane of first cleavage on the blastocyst
have proved more contentious (Piotrowska et al., 2001; Alarcon and Marikawa, 2003; Fujimori et al., 2003) are considered
elsewhere (Gardner, 2005).
499
R.L.Gardner and T.J.Davies
Rupture and retraction of the cortical granule envelope
that extends into the PVS from the outer surface of the vitellus
(Hoodbhoy et al., 2001) seems the most likely explanation
for the accumulation of a mass of granular material at the
cleavage plane in 2-cell conceptuses developing from
zygotes whose ZP was thinned with pronase. This envelope
forms in the PVS following fertilization and evidently makes
contact with the surface of the vitellus (Hoodbhoy et al.,
2001). How rapidly it is established is not clear, but is of
interest since off-axis first cleavage in zygotes without the
PVS gelated resulted less often in movement of the PB to the
cleavage plane in pronase-treated than untreated specimens.
This suggests that presence of an intact cortical granule envelope may facilitate centripetal movement of the PB, although
envisaging how it might do so depends on ascertaining
whether this envelope is established before or after the 2nd
PB forms. That this envelope may be important for normal
development is suggested by experiments in the hamster
where an inhibitory effect on cleavage was detected following its exposure to the lectin Concanavalin A or to polyclonal antisera raised against two of its constituent proteins (see
Hoodbhoy et al., 2001).
The present investigation has confirmed that the majority of
mouse zygotes are overtly bilaterally rather than radially symmetrical several hours before embarking on the first cleavage
division which is typically orthogonal to their bilateral plane.
While oocytes seem to require activation to become bilaterally
symmetrical morphologically, this change in overall shape evidently does not have to persist through to first cleavage for the
plane of this cleavage to be orthogonal to the bilateral plane
(Figures 4K, L and 5B). Hence, as indicated earlier, bilateral
symmetry may depend primarily on cytoplasmic organization
of the zygote that does not affect its overall form. This might
explain why not all zygotes show overt bilateral symmetry, and
raises the further question of whether unactivated oocytes
might also be ‘cryptically’ bilateral. These possibilities are difficult to explore unless bilateral organization proves to be discernible in living as opposed to fixed and stained specimens
which have so far provided the only evidence for its existence
in the mouse (reviewed in Dalcq, 1957; Gardner, 1996).
Regardless of the overall shape of bilateral zygotes, the plane
of bilateral symmetry was assumed to be meridional so that the oil
drops in the ZP were made to define a plane passing through the
zygote’s centre that was both parallel to its greater diameter and
included the 2nd PB. The validity of this assumption is underscored by the finding that even when first cleavage was obviously
non-meridional it was nevertheless orthogonal to the ringed plane.
The fact that cleavage is invariably orthogonal to the long axis of
the mitotic spindle in metazoan cells (Rappaport, 1986) means
that the spindle axis must lie in the ringed plane regardless of its
orientation relative to the zygote’s AV axis. Otherwise, where
cleavage was non-meridional its angle relative to the ringed plane
would decrease with increasing departure from the latter of the
plane in which the spindle lay. This is illustrated in Figure 8
which compares meridional versus non-meridional cleavage
when the bilateral and spindle planes either coincide or are at 45°
to each other. Hence, the choice of an AV plane as the bilateral
plane for ringing is clearly not arbitrary.
500
Figure 8. Diagrams showing that, even when not meridional, cleavage would be orthogonal to the animal–vegetal (AV) bilateral plane
only if the long axis of the mitotic spindle consistently lay in this plane.
(A, B, E, F) Zygotes viewed from the side along their bilateral plane;
(C, D, G, H) corresponding polar views. The bilateral plane is indicated by a continuous black line, the plane in which the long axis of the
mitotic spindle (red) lies is indicated by a dashed line or oval, the
cleavage plane by a green line or oval, while the second polar body
(2nd PB) is shown in blue. (A–D) In zygotes whose spindle and bilateral planes coincide, the cleavage plane is not only orthogonal to the
bilateral where cleavage is meridional (A, C) but also where it is not
(B, D). (E–H) In zygotes whose spindle plane does not coincide with
the bilateral plane, cleavage is orthogonal to the bilateral plane only
when cleavage is meridional (E, G) rather than non-meridional (F, H).
Not only is the orientation of first cleavage consistently related
to the zygote’s bilateral plane but, most interestingly, so are both
the Em.Ab axis and the bilateral plane of the blastocyst (Figure 9).
Bilateral symmetry of the mouse zygote
Figure 9. Summary of how the bilateral plane of the zygote (A), indicated by a dashed white line, maps on the 2-cell conceptus (B) and
early blastocyst (C, D). A–C are all in corresponding orientation with the second polar body centrally uppermost whilst D is rotated
through 90° so that the embryonic–abembryonic axis is vertical in order to show its bilateral plane.
However, while typically corresponding closely with each other
in orientation, the two bilateral planes were not always recorded
as being accurately parallel (Figure 7A and B). Obvious divergence of the Em.Ab axis of the blastocyst from parallel to the
zygote’s bilateral plane was apparent even in cases where the
2nd PB remained beneath its marker oil drops. While this suggests that such variability is real, the possibility that gelation of
the PVS might occasionally prevent the 2nd PB from rotating
within its ZP whilst allowing the conceptus to do so cannot be
discounted. Alternatively, it might be a consequence of variability in the pattern of subsequent cleavage (Gardner, 2002; Piotrowska-Nitsche et al., 2005). Obviously, other ways of
examining how the bilateral plane of the blastocyst relates to that
of the zygote are needed to clarify this important issue.
In conclusion, the present study shows that the majority of
PO and MF1 zygotes adopt a bilateral form several hours
before first cleavage. While this morphological change is contingent on activation of development, it could be an overt manifestation of intrinsic bilateral organization that already exists
prior to fertilization. That bilateral symmetry of the zygote is
of significance developmentally is evident from the fact that
the plane of first cleavage bears a strikingly consistent relationship to it. This is not true for other purported determinants of
this cleavage plane, namely the sperm entry point in conjunction with an entity closely associated with the PB (Plusa et al.,
2002a), or spatial relations between the sites of origin of the
two pronuclei (Hiiragi and Solter, 2004). That bilateral symmetry of the blastocyst relates to that of the zygote further
strengthens the case for dependence of early patterning on
processes that may be activated by the fertilizing sperm but
without regard to where the latter enters the oocyte.
Dependence of patterning during normal development
on cues that are present before cleavage raises questions
about the effectiveness with which regulative processes
operate if pre-patterning is perturbed. The fact that the
incidence of monozygotic twinning, particularly the late
originating monochorial monoamniotic type, is increased
significantly in human assisted conception (Alikani et al.,
2003) underlines the importance of elucidating the basis of
pre-patterning in mammals.
Acknowledgements
We wish to thank Andrea Kastner for help in preparing the manuscript
and both the Royal Society and the Biotechnology and Biosciences
Research Council for Support.
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Submitted on April 1, 2005; resubmitted on August 11, 2005; accepted on
August 22, 2005