Mammalian Development Laboratory, University of Oxford, Department of Zoology, South Parks Road, Oxford OX1 3PS, UK. E-mail: richard.gardner{at}zoology.ox.ac.uk
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Abstract |
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Key words: equatorial division/meridional division/mouse conceptuses/regular tetrahedral 4-cell/second cleavage
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Introduction |
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Although there is a consistent topographical relationship between the blastocyst and the 2-cell stage in the mouse (Gardner, 2001; Piotrowska et al., 2001
), how reproducibly the cytoplasm of the zygote is parcelled between blastomeres during cleavage is still contentious. Thus, first cleavage has been claimed both to be essentially random with respect to the animalvegetal axis of the zygote (Evsikov et al., 1994
), and almost invariably meridional (Howlett and Bolton, 1985
). In the mouse, as in many other eutherian mammals, second cleavage typically yields a crosswise arrangement of blastomeres so that the majority of 4-cell conceptuses approximate a regular tetrahedron in overall shape. Time-lapse studies in the rabbit led to the conclusion that, at least in this species, second cleavage is meridional for both 1/2 blastomeres, but that the animalvegetal axis of the second blastomere to divide rotates through nearly 90° before or during cytokinesis (Gulyas, 1975
). Limited observations in the mouse were said to be consistent with a similar pattern of second cleavage in this species (Gulyas, 1975
). More recently, in describing second cleavage in mammals, the term rotational has been interpreted to mean that one 1/2 blastomere divides meridionally and the other equatorially (Gilbert, 1997
). While no evidence was offered in support of this latter view, neither have the findings in the rabbit been shown to apply to other species. Moreover, although an approximately regular tetrahedral configuration of blastomeres with six intercellular contacts is the most common outcome of second cleavage in the mouse, other looser arrangements with between three and five such contacts also occur (Graham and Deussen, 1978
; Suzuki et al., 1995
).
Presently, therefore, it is not possible to assess the extent to which partitioning of the cytoplasm of the zygote varies between conceptuses during early cleavage in the mouse. This is particularly true for second cleavage where, as indicated above, it is still uncertain how the division planes of sister 1/2 blastomeres relate to the animalvegetal axis of the zygote. One obstacle to solving this problem has been the seeming lack of an enduring marker for this axis because the most obvious candidate, the second polar body, was claimed to be freely motile during cleavage (Lewis and Wright, 1935; Borghese and Cassini, 1963
). However, it is now evident that this polar body is tethered at its site of production, and thus continues to mark the animal pole of the zygote for as long as it survives (Gardner, 1997
). This finding has been exploited here to re-examine the orientation of cytokinesis with respect to the animalvegetal axis in the blastomeres of 2-cell conceptuses that yield 4-cell stages of the predominant, approximately regular, tetrahedral form. Such regular tetrahedral 4-cell conceptuses were found to result from meridional division of one 1/2 blastomere and approximately equatorial division of the other. While the products of the meridional division could not be distinguished from each other, those of the equatorial division were readily differentiated by virtue of their differing proximity to the 2nd polar body. This means that regular tetrahedral 4-cell stage mouse conceptuses are composed of blastomeres that can be assigned to three distinct categories according to their endowment with cytoplasm from different axial levels of the zygote.
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Materials and methods |
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Conceptuses whose further development was to be tested in vivo following manipulation were cultured overnight before being transferred to the oviducts of anaesthetized recipients during the morning of the first day of pseudopregnancy.
Blastomeres of 2- and 4-cell conceptuses are denoted as 1/2 and 1/4 blastomeres respectively in accordance with the standard convention (Tarkowski and Wroblewska, 1967).
Vegetal polar labelling of 1/2 blastomeres
For this, the surface of one or, in some cases, both blastomeres was decorated locally with fluorescent latex microspheres (Fluoresbrite yellow-green carboxylate; Polysciences, Warrington, PA, USA). Following various trials, satisfactory labelling was achieved by selectively exposing the vegetal polar region of 1/2 blastomeres to microspheres after it had been exteriorized by applying gentle suction via a flame-polished micropipette to an appropriately sited hairline slit in the zona pellucida (Tsunoda et al., 1986). Microspheres suspended in low protein medium adhered rapidly and firmly to the surface of the exteriorized region. After brief exposure to the suspension, conceptuses were rinsed thoroughly in fresh KSOMHEPES to eliminate all unattached microspheres before the exteriorized blastomere region was gently pushed back inside the zona pellucida with a blunt glass probe. Approximately 0.20 µm diameter microspheres were used in an initial series of experiments. However, these proved too small for accurately circumscribed labelling because of the ease with which they were endocytosed. Hence, only Fluoresbrite microspheres from a batch with a mean diameter of ~0.5 µm that were retained by a 0.45 µm filter (Millipore Corp., Bedford, MA, USA) were adopted thereafter. For labelling sister blastomeres, the vegetal polar regions of both were either externalized simultaneously via a single slit in the zona pellucida or successively via two separate ones. Following microsphere labelling, one 1/2 blastomere was labelled with 1,1-dioctadecyl-3-3-3'-3'-tetramethylindocarbocyanine perchlorate (DiI; Molecular Probes Inc, Eugene, Oregon, USA) before the conceptuses were cultured through second cleavage so as to allow sister pairs of 1/4 blastomeres to be distinguished thereafter. The dye was made up as a saturated stock in absolute ethanol that was then diluted 1/5 with either soya or olive oil (Gardner, 1997
; Gardner and Davies, 2002
). Its use also enabled the occurrence of ectopic microspheres to be monitored in all experiments in which only one 1/2 blastomere was exposed to them.
Gelation of the perivitelline space
Conceptuses recovered at an advanced 2-cell stage were incubated for 8090 min in pre-equilibrated KSOM medium containing the sodium salt of alginic acid (low viscosity; Sigma, Poole, UK) at 1% (w/v), as described elsewhere (Davies and Gardner, 2002). Thereafter, they were rinsed briefly in KSOMHEPES before being incubated for 30 min at room temperature in a solution of 0.9% NaCl plus 1.5% CaCl2·2H2O diluted 1/9 with KSOMHEPES. After further rinsing in KSOMHEPES following gelation of the perivitelline space, conceptuses were placed in a micromanipulation chamber so that the location of the second polar body could be marked by injecting a small drop of oil into the immediately overlying region of the zona pellucida (Gardner, 2001
). Finally, they were rinsed and then incubated in pre-equilibrated KSOM minus alginate for scoring at the 4-cell stage. The site of the second polar body was also marked with oil in the zona pellucida in additional 2-cell conceptuses that had not been exposed to alginate.
Dissociation of 2-cell conceptuses and destruction of sister pairs of 1/4 blastomeres
For separating viable sister 1/2 blastomeres after both had had their vegetal polar region labelled with microspheres, conceptuses were incubated at 37°C in calcium- and magnesium-free OC medium containing 0.02% EGTA (Sigma) for up to 20 min following dissolution of the zona pellucida with acidified Tyrodes saline (Gardner and Davies, 2000). They were then transferred to KSOMHEPES for dissociation by repeated gentle aspiration using a pipette with a flame-polished aperture that was about two-thirds of their diameter. Sister 1/4 blastomeres were destroyed in situ by repeatedly stabbing and tearing them with a sharp-tipped glass micropipette inserted through the zona pellucida whilst the conceptuses were immobilized on the tip of a holding pipette. They were identified by prior ionophoretic injection of a 4% solution of Fluorescein Complexon (Eastman Kodak Co., Rochester, NY, USA) in 0.1 mol/l KCl into one blastomere (Gardner, 1997
).
Disrupting the intercellular bridge between sister 1/4 blastomeres
Sister 1/4 blastomeres were identified at the 3-cell stage when both were marked by intracellular injection of a drop of silicone oil (MS 550; BDH, Poole, UK) before the conceptuses were cultured to the 4-cell stage. For reducing the adhesion between blastomeres, conceptuses were incubated for up to 30 min in calcium- and magnesium-free OC medium containing EGTA. Blastomeres were exteriorized by applying gentle suction to them via a micropipette after a lengthy hairline slit had been made in the overlying zona pellucida. They were then replaced inside the zona pellucida with a micropipette, either whilst still connected to their sister, or after the connection had been severed with a glass needle. Some conceptuses recovered at the 4-cell stage were reconstructed following complete dissociation. For this, the zona pellucida was slit after they had been incubated in calcium- and magnesium-free OC medium containing EGTA. Repeated aspiration with a flame-polished pipette was then used to dissociate them completely, after which the separated blastomeres were replaced inside the evacuated zona pellucida with a micropipette.
Distinguishing polar bodies
Throughout this study, the animal pole was taken as the site of the second polar body. Therefore, it was important to be able to distinguish this body reliably from the first polar body which not infrequently persists during early cleavage in the PO strain, particularly as the two bodies often lie well apart (Gardner, 2002). Through division of the first polar body and occasional deutoplasmolysis (Dalcq, 1957
) three or more candidate polar bodies can be present. While the second polar body is typically nucleated and the first polar body (and products of deutoplasmolysis) not (Longo, 1987
), such a distinction is often not clear by differential interference contrast microscopy. It could usually be made by epifluorescence microscopy after incubating conceptuses for 10 min at 37°C in medium containing Hoechst 33342 (Sigma, Poole, UK) at 1 µg/ml. However, such staining was considered appropriate only for conceptuses that did not require further culture prior to scoring. Where further culture was required, all conceptuses for which the identity of the second polar body was uncertain were discarded. Finally, unless otherwise qualified, hereafter the abbreviation PB refers specifically to the second polar body.
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Results |
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Overall, >80% of 2-cell conceptuses proved suitable for vegetal polar labelling of one or both blastomeres. Thereafter, one blastomere was also labelled with DiI before the conceptuses were cultured to the 4-cell stage. Where the vegetal polar region of only one blastomere had been decorated with microspheres, DiI labelling was invariably applied to the other blastomere. Once the double-labelled 2-cell conceptuses had completed second cleavage, they were re-examined to check their suitability for scoring the distribution of the microspheres. Here, as discussed earlier, only regular tetrahedral 4-cell conceptuses with the features shown in Figure 1 were selected for detailed scoring. The data for labelling of one 1/2 blastomere of 2-cell conceptuses with microspheres of ~0.20 µm versus >0.45 µm in diameter are presented in Table II
. It will be seen that, in the great majority of resulting regular tetrahedral 4-cell conceptuses, the microspheres were either shared between two of the blastomeres adjacent to the PB that were identifiable as sisters from the distribution of DiI, or confined to the PB-remote sister of the third such blastomere (Table II
; Figure 5A, B
). In addition, in each of three cases where the vegetal polar region of both 1/2 blastomeres was labelled, the sister of the PB-remote blastomere was the only one devoid of microspheres at the 4-cell stage (data not shown).
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In the scheme of second cleavage proposed for the rabbit (Gulyas, 1975), the second 1/2 blastomere to divide is invariably the one that undergoes axial rotation. Hence, if the mouse accords with the rabbit, the 1/2 blastomere showing an equatorial cleavage plane should always be the second to divide. This was checked by recording the frequency with which the cleavage plane of sister 2/4 blastomeres was approximately parallel versus orthogonal to the animalvegetal axis in 3-cell conceptuses with an intact unambiguous PB. Among 81 such conceptuses entering second cleavage in vitro, the relationship of the 2/4 pair to the PB was consistent with their being the product of meridional division in 49 (60%), and of equatorial division in the remaining 32 (40%). Among 48 conceptuses that were recovered from in vivo at the 3-cell stage, the 2/4 pair of blastomeres appeared to be the product of meridional versus equatorial division in 23 (48%) and 25 (52%) cases respectively. Given that not all 4-cell conceptuses are regular tetrahedrons, meridional orientation of the cleavage plane in the first 1/2 blastomere to divide does not invariably lead to equatorial orientation in the second and vice versa. Indeed, where orientation of cleavage of the second 1/2 blastomere to divide was also recorded, in several cases it was found to be the same as the first. Moreover, in additional 3-cell stages, the cleavage plane of the 2/4 pair of blastomeres could not be classified as either meridional or equatorial, but was clearly intermediate between the two. Indeed, the form of a substantial proportion of the minority of 4-cell conceptuses that could not be classified as regular tetrahedrons was consistent with oblique cleavage of one or both 1/2 blastomeres.
Orientation of cytokinesis in separated sister 1/2 blastomeres
While the foregoing findings show that the two distinct orientations of cytokinesis yielding regular tetrahedral 4-cell conceptuses are not determined by division order, they might nevertheless be interdependent. This possibility was explored by culturing sister 1/2 blastomeres separately after the vegetal polar region of each had been decorated with microspheres. To ensure that isolation was done well before second cleavage, only 2-cell conceptuses in which both blastomeres had intact nuclei with prominent nucleoli were used in these experiments. From the data presented in Table III, it can be seen that very unequal labelling of members of one or both sister 2/4 pairs was encountered in a high proportion of cases using the smaller microspheres. This may be because these were much more readily endocytosed than the larger ones and, given the likelihood of delayed division of isolated blastomeres, could have had time to become well-dispersed within the cytoplasm before cytokinesis. Nevertheless, results consistent with one sister 1/2 blastomere dividing meridionally and the other equatorially were obtained using both smaller and larger microspheres (see Table III
). Examples of meridional versus equatorial division of isolated 1/2 blastomeres are shown in Figure 5C and D
.
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Blastomere relations in regular tetrahedral 4-cell conceptuses
According to the present findings, the majority of 4-cell conceptuses have a regular tetrahedral shape because the cleavage plane of one 1/2 blastomere is usually parallel to, and that of the other approximately orthogonal to, the animalvegetal axis. One product of the equatorial division can be identified reliably because, unlike both its sister and the products of the meridional division, it is remote from, rather than adjacent to, the PB (see Figure 1). Detailed microscopic examination of further living regular tetrahedral 4-cell conceptuses was undertaken to address two questions. First, can the PB-adjacent sister of the PB-remote blastomere be distinguished from the products of the meridional division without recourse to lineage-labelling? Second, is it possible to differentiate reliably between the products of the meridional division? A way of approaching both questions has been suggested in pioneering lineage studies (Graham and Deussen, 1978
). These workers noted that when regular tetrahedral 4-cell conceptuses were oriented with three blastomeres in the same plane, the three did not overlap the overlying fourth blastomere to the same extent. Specifically, sister blastomeres seemed to overlap each other more than non-sisters.
Hence, regular tetrahedral conceptuses were oriented on the tip of a holding pipette so that the three PB-adjacent blastomeres were co-planar beneath the fourth, PB-remote blastomere. This was done at high magnification and with the condenser diaphragm opened widely so as to minimize the depth of focus and thus ensure accuracy of orientation. Reorientation and re-scoring were undertaken wherever differences in overlap appeared marginal. Cases where one PB-adjacent blastomere overlapped the PB-remote one to a greater extent than did either of the other two were no more common than cases where two PB-adjacent blastomeres overlapped it to a similar extent. Much more often, one PB-adjacent blastomere overlapped the PB-remote one to a lesser extent than either of the other two. By DiI-labelling, this blastomere was found to be the sister of the PB-remote blastomere in only 3/41 cases (7%). In a further series of regular tetrahedral conceptuses, the PB-remote blastomere was labelled with DiI in order to identify the products of the meridional division and ascertain whether they differed consistently in the extent to which they overlapped it (see Figure 8). Among a total of 28 regular tetrahedral 4-cell conceptuses that were examined thus, both products of the meridional division overlapped the PB-remote blastomere to a similar extent in six. In the remaining 22 cases where one product clearly showed less overlap than the other, this was the more clockwise of the two in 13 cases and the less clockwise in nine (Figure 8
). Specimens depicting this variability are shown in Figure 9
.
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Discussion |
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According to Gulyas (1975), it is invariably the second 1/2 blastomere to divide that undergoes axial rotation and should thus correspond to the equatorially dividing blastomere in the present study. However, in the mouse, an equatorial orientation of cytokinesis was seen about as often in the first as in the second 1/2 blastomere to divide, regardless of whether second cleavage was initiated in vivo or in vitro. Furthermore, comparison of the orientation of cytokinesis in sister 1/2 blastomeres that had been separated well before the onset of second cleavage argues against interdependence of the two patterns of division. Although the frequency with which one sister showed meridional and the other equatorial division was low compared to that seen in intact 2-cell conceptuses, it still rivalled that in intact specimens recovered at the 3-cell stage. However, while these data on the division of separated 1/2 blastomeres clearly show that there is no default orientation of the division plane in second cleavage, they are too limited to decide whether the choice for a given blastomere might be pre-programmed rather than random.
Collectively, the present findings support the conclusion that the typical regular tetrahedral form of the 4-cell mouse conceptus is due to meridional division of one 1/2 blastomere and approximately equatorial division of the other. This means that three blastomeres are normally in contact with the PB and the fourth remote from it (Figures 1, 9 and 10). Consequently, 1/4 blastomeres can be assigned to three categories according to how much of the animalvegetal axis of the zygote they inherit. Thus, while both products of meridional division acquire the entire axis, one product of the equatorial division is endowed essentially with only its animal, and the other with only its vegetal, half. For convenience, the products of the meridional division are referred to hereafter as the A/B pair, and the PB-adjacent and PB-remote products of the equatorial division as C and D blastomeres, respectively (Figure 10
). It should be noted that this differs from previous use of such blastomere notation (Gulyas, 1975
; Graham and Deussen, 1978
; Kelly et al., 1978
) in that it relates to the orientation rather than the order of division of 1/2 blastomeres. Attempts to distinguish reliably between members of the A/B pair of blastomeres according to their spatial relationship with D were unsuccessful.
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It is noteworthy that nearly one-third of conceptuses whose second cleavage took place in vivo did not conform to the regular tetrahedral shape, a consistently higher proportion than among those that developed from the 2- to the 4-cell stage in vitro. This might be because the substantial compression which conceptuses evidently endure during their passage through the oviduct (Nichols and Gardner, 1989) can perturb the orientation of cytokinesis. Regardless, the non-regular tetrahedral conceptuses included essentially planar forms that seem to have resulted from either meridional or equatorial division of both 1/2 blastomeres (Figure 6
). The incidence of non-regular tetrahedral conceptuses is too high to allow for the possibility that regular second cleavage is prerequisite for normal development. Nonetheless, there are indications that blastulation may be delayed and the rate of postimplantation development possibly reduced in 4-cell stage conceptuses with fewer than five or six intercellular contacts (Graham and Deussen, 1978
; Suzuki et al., 1995
).
Recent studies suggest that in undisturbed development the equator of the blastocyst corresponds with the plane of first cleavage (Gardner, 2001; Piotrowska and Zernicka-Goetz, 2001
). Accordingly, the progeny of one 1/2 blastomere might be expected to form the polar trophectoderm and deeper cells of the inner cell mass and the other the mural trophectoderm plus the more superficial cells of the inner cell mass. However, in an elegant analysis of lineage from the 2-cell stage in which both 1/2 blastomeres were labelled differentially (Piotrowska et al., 2001
), the boundary between the clones was found to be rather variable and clearly not strictly orthogonal to the embryonicabembryonic axis of the blastocyst. Such variability is readily explicable in the light of the present findings since development of the blastocysts that were analysed would have proceeded via a range of different patterns of second cleavage. Nonetheless, overall, the analysis should reflect lineage that proceeded via the predominant regular tetrahedral configuration of blastomeres at the 4-cell stage. A notable feature of this configuration is that the D blastomere caps the vegetal polar region asymmetrically instead of lying wholly to one side of it, thereby distorting the originally straight boundary set by the plane of first cleavage (Figure 10B
). Clonal descendants of this blastomere would therefore be expected to span both hemispheres of the blastocyst on the side opposite the PB. Preliminary findings on the distribution of clonal descendants of the D blastomere at the early blastocyst stage accord with this expectation (G.Bressan, T.J.Davies and R.L.Gardner, unpublished observations).
Selecting conceptuses with a common pattern of cleavage is important for minimizing variability and thus enhancing the ratio of signal to noise in investigating both the normal fate of blastomeres and their developmental potential. While the demonstration that a substantial majority of conceptuses have a regular pattern of second cleavage is most encouraging, answers to further questions are needed before it is possible to decide whether this goal is attainable. For example, does regularity of second cleavage simply depend on whether first cleavage is also regular, i.e. accurately meridional, or are other factors involved? Does it also guarantee a consistent pattern of third cleavage?
Finally, it is important to note that while the regular tetrahedral pattern of second cleavage exhibited by more than two-thirds of conceptuses results in consistent partitioning of cytoplasm of the zygote in relation to its animalvegetal axis, the possibility remains that there are developmentally significant asymmetries orthogonal to this axis. If the latter proved to be the case, it would be important to know whether or not these also bear a consistent relationship to early cleavage planes.
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Acknowledgements |
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Submitted on June 19, 2002; accepted on August 8, 2002.