Experimental analysis of second cleavage in the mouse

R.L. Gardner

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Mammalian conceptuses typically have an approximately regular tetrahedral shape at the 4-cell stage. In the rabbit, this has been attributed to both 2-cell blastomeres dividing meridionally, but with the animal–vegetal axis of the second blastomere to divide rotating through roughly 90° before or during cytokinesis. The aim of the present study was to ascertain whether this was also true for the mouse. METHODS AND RESULTS: First, the distribution in regular tetrahedral 4-cell conceptuses of fluorescent microspheres applied to the vegetal polar region of one or both blastomeres at the 2-cell stage was analysed. Second, the ability of 2-cell stages to form regular tetrahedral 4-cell conceptuses after the previtelline space had been gelated to prevent blastomeres from rotating was also investigated. Neither experiment yielded evidence supporting blastomere rotation during second cleavage. Rather, the findings were consistent with the regular tetrahedral form of 4-cell conceptus resulting from meridional division of one blastomere and approximately equatorial division of the other. CONCLUSIONS: Second cleavage in the mouse typically yields 4-cell conceptuses with three distinct types of blastomere. While both products of the meridional division include all axial levels of the zygote, those of the equatorial division acquire only its vegetal or animal half.

Key words: equatorial division/meridional division/mouse conceptuses/regular tetrahedral 4-cell/second cleavage


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Various findings conflict with the long-cherished notion that patterning during early development in mammals cannot depend on information that is already established within the zygote (Gardner, 1996Go ,1998Go; Gardner and Davies, 2000Go). Particularly challenging is recent evidence that the axes of the blastocyst, which have been implicated in specifying those of the fetus (Smith, 1980Go ,1985Go; Gardner et al., 1992Go; Weber et al., 1999Go), are already specified by the onset of cleavage in the mouse (Gardner, 2001Go; Piotrowska and Zernicka-Goetz, 2001Go; Piotrowska et al., 2001Go). However, a claim that this early patterning is related to the site of sperm entry (Piotrowska and Zernicka-Goetz, 2001Go; Plusa et al., 2002Go) is not supported by detailed examination of the distribution of sperm components (Davies and Gardner, 2002Go). The relevant patterning information would therefore seem to be intrinsic to the egg. Hence, investigating how the cytoplasm of the egg is partitioned during cleavage might offer insight into its role in patterning the early embryo. If partitioning proved to be regular, the significance of different cytoplasmic regions could be assessed by testing the developmental potential of the blastomeres that acquire them.

Although there is a consistent topographical relationship between the blastocyst and the 2-cell stage in the mouse (Gardner, 2001Go; Piotrowska et al., 2001Go), 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 animal–vegetal axis of the zygote (Evsikov et al., 1994Go), and almost invariably meridional (Howlett and Bolton, 1985Go). 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 animal–vegetal axis of the second blastomere to divide rotates through nearly 90° before or during cytokinesis (Gulyas, 1975Go). Limited observations in the mouse were said to be consistent with a similar pattern of second cleavage in this species (Gulyas, 1975Go). 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, 1997Go). 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, 1978Go; Suzuki et al., 1995Go).

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 animal–vegetal 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, 1935Go; Borghese and Cassini, 1963Go). 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, 1997Go). This finding has been exploited here to re-examine the orientation of cytokinesis with respect to the animal–vegetal 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Recovery, culture and transfer of conceptuses
Albino mice of the closed-bred PO strain were used throughout the study, some being exposed to darkness between 19:00 and 05:00 (normal), and the remainder between 13:00 and 21:00 (altered). Females were selected for estrus by external inspection (Champlin et al., 1973Go), paired with fertile or vasectomized males before onset of the dark period, and checked for a vaginal plug the following morning. Fertile-mated females on the normal and altered light regimes were killed for recovery of 2-cell conceptuses in the afternoon and morning respectively of the second day of pregnancy. The excised oviducts were flushed with potassium-enriched synthetic oviductal medium (KSOM)–HEPES (Summers et al., 1995Go) using a fine-tipped Pasteur pipette inserted into one or other end, and conceptuses were kept in this medium both before and during their micromanipulation. For culture, conceptuses were transferred to drops of KSOM medium plus amino acids (Summers et al., 2000Go) under light paraffin oil in small bacteriological dishes in which they were incubated at 37°C in an atmosphere of 5% CO2 in air.

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, 1967Go).

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., 1986Go). 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 KSOM–HEPES 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, 1997Go; Gardner and Davies, 2002Go). 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 80–90 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, 2002Go). Thereafter, they were rinsed briefly in KSOM–HEPES 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 KSOM–HEPES. After further rinsing in KSOM–HEPES 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, 2001Go). 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 Tyrode’s saline (Gardner and Davies, 2000Go). They were then transferred to KSOM–HEPES 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, 1997Go).

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, 2002Go). Through division of the first polar body and occasional deutoplasmolysis (Dalcq, 1957Go) 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, 1987Go), 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.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Form and frequency of regular tetrahedral 4-cell conceptuses
Four-cell conceptuses were classified as being regular tetrahedrons if they had six approximately equal intercellular contacts (Graham and Deussen, 1978Go; Suzuki et al., 1995Go), and therefore approached 4-fold axes of symmetry in the disposition of their blastomeres. Two typical examples of such 4-cell conceptuses are shown with their animal–vegetal axis both vertical and horizontal in Figure 1Go. The proportion of regular tetrahedral conceptuses among 4-cell stages developing in vivo was 228/324 (70%). The corresponding figures were 94/110 (85%) for non-manipulated conceptuses that went through second cleavage in vitro following explantation at the 2-cell stage, and 11/25 (44%) for those recovered at the 3-cell stage that completed second cleavage in vitro. However, not all regular tetrahedral 4-cell conceptuses were suitable for analysing the orientation of second cleavage planes with respect to the animal–vegetal axis of the zygote. This requires that the PB should not only be intact but, to avoid cases of markedly non-meridional first cleavage, lies by the intersection between three blastomeres (Figure 1Go). These two requirements were met in 87% of 245 regular tetrahedral 4-cell conceptuses that were examined in detail.



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Figure 1. (AC) Photographs of a regular tetrahedral 4-cell conceptus with its animal–vegetal axis vertical (A, B) versus horizontal (C). In A, focus is on the animal pole to show that the PB lies symmetrically over the intersection between three blastomeres, and in B on mid-plane to show the location of the PB-remote, fourth blastomere. (DF) Views corresponding to AC of a second example of a regular tetrahedral 4-cell conceptus in which the PB-remote blastomere is less off-centre with respect to the other three. The site of the PB is indicated by an arrow in C and F.

 
Identifying sister pairs of 1/4 blastomeres
If 4-cell conceptuses are cultured for 1.5–2 h following labelling of one 1/4 blastomere with DiI, this fluorescent dye almost invariably spreads just to one other blastomere. This seemed most likely to be due to persistence between sister 1/4 blastomeres of the intercellular bridge formed during second cleavage (Goodall and Johnson, 1984Go) (Figure 2A, BGo). In view of the importance of being able to distinguish sister blastomeres reliably in the experiments that follow, it was necessary to confirm that this was indeed the case. To this end, both 1/4 blastomeres of 3-cell conceptuses obtained directly from the oviduct or through short-term culture of late 2-cell stages were injected with a drop of silicone oil (Figure 2CGo). The conceptuses were then cultured to the 4-cell stage when some, without further manipulation, had either one of the oil-marked or the unmarked blastomeres labelled with DiI. The remainder were incubated in calcium- and magnesium-free OC medium with EGTA before a slit was made in the zona pellucida through which one of the marked or unmarked blastomeres was exteriorized. In some cases this blastomere was replaced inside the zona pellucida whilst it was still connected to its sister and, in others, after the connection had been broken. Thereafter, either the exteriorized blastomere or its sister was labelled with DiI. All conceptuses were examined by fluorescence microscopy after further culture for up to 2 h following DiI labelling. The results, which are presented in Table IGo, show that the DiI spread in all but three instances where blastomeres remained connected, and invariably did so obviously only to the sister of the one that was labelled. They also show that the dye never spread between sister blastomeres once they had been separated, despite their regaining extensive contact thereafter (Table IGo and Figure 3Go). Moreover, in 4-cell conceptuses that were reconstructed within an evacuated zona pellucida following complete dissociation, DiI invariably remained confined to whichever 1/4 blastomere was labelled (Table IGo). Collectively, these findings confirm that labelling of one blastomere with DiI is a valid way of identifying its sister.



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Figure 2. (A, B) Two examples of sister pairs of 1/4 blastomeres that have been drawn apart with needles following incubation in calcium- and magnesium-free OC medium with EGTA to show that they remain connected by a prominent intercellular bridge whose mid-body is particularly clearly demarcated (arrow) in B. (C) Side view of a 3-cell stage conceptus at lower magnification in which the 2/4 pair is the product of an equatorial as opposed to a meridional division. Marking both 1/4 blastomeres with an oil drop at this stage enabled sister pairs to be identified unequivocally following completion of second cleavage.

 

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Table I. Spread of DiI between blastomeres at the 4-cell stage
 


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Figure 3. Spread versus failure of spread of DiI in 4-cell conceptuses according to whether the intercellular bridge linking the labelled blastomere with its sister was disrupted (AD) or left intact prior to labelling (EH). A persisting remnant of the bridge is arrowed in B and D. (A, C, E, G) Brightfield images; (B, D, F, H) epifluorescence images. The location of the slit in the zona pellucida is marked with a black arrow in A, C, E and G.

 
Polar labelling of one or both 1/2 blastomeres
The vegetal rather than animal pole of blastomeres was chosen for labelling with fluorescent microspheres in order to minimize the risk of damaging or displacing the PB which was used to determine the orientation of the animal–vegetal axis. The rationale of this approach was that if both 1/2 blastomeres undergo meridional division, as has been asserted for the rabbit (Gulyas, 1975Go), this should invariably result in daughter pairs of 1/4 blastomeres being labelled in their region of apposition (Figure 4A–CGo). If, however, one blastomere engages in meridional and the other in equatorial division, the label should as often be associated with only one of the pair of resulting daughter 1/4 blastomeres as with both (Figure 4D–FGo). Moreover, where the label partitions to one daughter, this should be the 1/4 blastomere that is remote from the PB, and where it is distributed to both, both should be adjacent to this body (Figure 4FGo).



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Figure 4. Diagrams illustrating two alternative schemes for second cleavage that could account for the predominant crosswise or regular tetrahedral form of the 4-cell stage, and expected differences in the distribution of fluorescent microspheres after labelling the vegetal polar region of 1/2 blastomeres. Orientation of the animal–vegetal axis in individual 1/2 blastomeres is indicated by a dashed white line and their cleavage plane by a continuous black line. (AC) Scheme according to Gulyas (1975) in which the animal–vegetal axis of the second 1/2 blastomere to divide rotates ~90° before or during cytokinesis. All four 1/4 blastomeres should carry microspheres following vegetal polar labelling of both 1/2 blastomeres and both members of one sister pair of 1/4 blastomeres should always do so after labelling one 1/2 blastomere. (DF) Scheme according to Gilbert (1997) in which one 1/2 blastomere divides meridionally and the other approximately equatorially. In this case, the microspheres should be distributed to both products of the 1/2 blastomere that divides meridionally and to only the PB-remote (= vegetal) product of the 1/2 blastomere that divides equatorially.

 
In practice, not all 2-cell conceptuses were suitable for labelling because of uncertainty about the orientation of their animal–vegetal axis. While, occasionally, this was due to the lack of an intact PB or because the plane of first cleavage was patently off-axis, more commonly up to three or more candidate bodies could be present which were not always adjacent to each other. Use of Hoechst 33342 to differentiate between the latter (Gardner, 1997Go) was avoided in case exposing conceptuses to fluorescence microscopy perturbed second cleavage. Hence, except where the first PB could be recognized unambiguously through having undergone partial or complete division, only 2-cell conceptuses with a single intact PB were selected for labelling. According to previous experience with Hoechst staining, where just one intact PB was present, its staining pattern was that expected of the second rather than the first (Longo, 1987Go) in 98% of conceptuses (Gardner, 1997Go).

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 1Go 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 IIGo. 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 IIGo; Figure 5A, BGo). 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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Table II. Distribution of microspheres at the 4-cell stage following in-situ labelling of the vegetal pole of one blastomere at the 2-cell stage
 


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Figure 5. (A, B) Typical examples of fluorescent microsphere distribution following second cleavage of specimens in which the vegetal polar region of one 1/2 blastomere had been labelled. The location of the PB is indicated by a white arrow. (A) Two of the three PB-adjacent blastomeres are labelled so that their division plane must have been meridional. (B) Only the PB-remote blastomere is labelled so that the division plane must have been away from the vegetal polar region. (C, D) Pairs of sister 1/4 blastomeres obtained from 1/2 blastomeres that were isolated following vegetal polar labelling with flurorescent microspheres. (C) A case where the cleavage plane must have bisected the vegetal polar region versus (D) where it must have been nearly orthogonal to it.

 
In the foregoing analysis, only conceptuses that formed regular tetrahedral 4-cell stages following vegetal polar labelling, which constituted the great majority (see Table IIGo, columns 2 and 3), were examined in detail. Both the distribution of the microspheres and the relationship between blastomeres varied markedly in the remainder. However, included among the latter were specimens that were essentially planar, with the PB lying either on the middle of one side of the plane by the common boundary between all four blastomeres (Figure 6A, BGo), or at one end between just two of them (Figure 6C, DGo). Nonetheless, second cleavage seems most commonly to entail meridional division of one 1/2 blastomere and non-meridional or approximately equatorial division of the other.



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Figure 6. Two categories of essentially planar (four intercellular contact) 4-cell conceptuses with the PB either at the middle ofone side (A, B) or at one edge between two blastomeres (C, D).(C) Brightfield image with the PB in focus and a divided1st PB plus deutoplasmolytic body lying out of focus beneath.(D) Epifluorescence image of (C) identifying sister pairs through the spread of DiI. Note that this is one of the rare cases where DiI spread both to the sister of the labelled blastomere and, much more weakly, also to the other pair of blastomeres. The relationship of the strongly and weakly labelled 2/4 pairs to the PB argues that both were products of equatorial division.

 
Second cleavage following gelation of the perivitelline space
If the predominant regular tetrahedral form of the 4-cell conceptus is due to the animal–vegetal axis of one 1/2 blastomere rotating through nearly 90° before or during cytokinesis, this form should not occur if such rotation is prevented. Advanced 2-cell conceptuses were incubated for up to 1.5 h in medium containing low viscosity sodium alginate. Following a brief rinse in alginate-free medium, they were exposed to elevated calcium so as to gelate the perivitelline space before the locus of the PB was marked with small oil drops in the zona pellucida. Both specimens with the perivitelline space gelated, and controls that had not been exposed to sodium alginate were then cultured through second cleavage so that the incidence of early 4-cell stages with a regular tetrahedral shape could be compared. It was found that conceptuses with a gelated perivitelline space in which the PB remained well-aligned with its marker oil following 2nd cleavage could have six cell contacts with the PB adjacent to three blastomeres and remote from the fourth. However, such conceptuses appeared somewhat compacted (cf. Figures 7 and 1GoGo), as might be expected from their being surrounded by a layer of gel. Nonetheless, orientation of the two cleavage planes relative to the PB in the alginate-treated conceptuses was consistent with meridional division of one 1/2 blastomere and equatorial division of the other in 18/30 (60%) cases compared with 15/25 (60%) in the controls. Hence, preventing blastomere rotation during second cleavage evidently does not suppress formation of the predominant regular tetrahedral form of 4-cell conceptus.



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Figure 7. Differential interference contrast images of examples of regular tetrahedral 4-cell conceptuses developing from the 2-cell stage with the perivitelline space gelated showing that one 1/2 blastomere still had its division plane roughly orthogonal to the animal vegetal axis. (A) Specimen with its animal–vegetal axis horizontal and the first cleavage plane vertical, in which one product of the meridional division (left) lies directly beneath the other. Note the approximately E division plane of the other (right) sister pair of blastomeres (arrow). (B, C) A second specimen with both its animal–vegetal axis and first cleavage plane approximately horizontal. Focus is on the upper pair of blastomeres with an equatorial division plane (arrowed) in (B) and on the lower pair with a meridional division plane in (C) which also shows the PB. Note that in both specimens contact between blastomeres is more extensive than in early regular tetrahedral 4-cell conceptuses whose pervitelline space had not been gelated (cf. Figures 1, 5 and 9GoGoGo).

 
PB attachment, and division order of 1/2 blastomeres
In the foregoing experiments, lack of displacement of the PB relative to its oil marker in the zona pellucida was taken as evidence that gelation of the perivitelline space had precluded blastomere rotation. However, this would not be valid if, according to one proposed scheme of second cleavage (Gulyas, 1975Go), the PB was invariably attached to the 1/2 blastomere whose animal–vegetal axis does not rotate. Hence, further regular tetrahedral 4-cell conceptuses were divested of the zona pellucida with acidified Tyrode’s saline so as to enable the blastomere to which the PB was attached to be ascertained by gentle probing with a pair of blunt-tipped glass needles (Gardner, 1997Go). Prior labelling with DiI was used to enable sister 1/4 blastomeres to be distinguished from non-sisters. Among a total of 60 regular tetrahedral 4-cells, the PB was found to be attached to a daughter of the 1/2 blastomere that had divided meridionally in 37 (62%), and to a daughter of the one that had divided equatorially in the remaining 23 (38%). With this information to hand, additional experiments were undertaken in which the PB was marked with oil drops in the zona pellucida at the late 2-cell stage without either gelating the perivitelline space or exposing the conceptuses to any further manipulation. Following such strictly non-invasive marking (Gardner, 2001Go), the conceptuses were cultured through second cleavage for rechecking the relationship between PB and its marker oil at the early 4-cell stage. A disparity consistent with a >=70° rotation of the animal–vegetal axis of one blastomere was observed in only 4/49 conceptuses that formed regular tetrahedral 4-cell stages. This is significantly less than the frequency with which the PB was found to be attached to the 1/2 blastomere that divided equatorially ({chi}2 = 11.60, P < 0.002).

In the scheme of second cleavage proposed for the rabbit (Gulyas, 1975Go), 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 animal–vegetal 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 IIIGo, 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 IIIGo). Examples of meridional versus equatorial division of isolated 1/2 blastomeres are shown in Figure 5C and DGo.


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Table III. Distribution of microspheres between daughter blastomeres following cleavage of vegetally labelled, separated sister pairs of 1/2 blastomeres
 
Analysis of the developmental potential of sister pairs of 4-cell blastomeres
A small series of experiments was undertaken to compare the developmental potential of the 2/4 products of meridional versus equatorial second cleavage divisions. For this, fluorescein complexon was injected ionophoretically into one of the three PB-adjacent blastomeres of regular tetrahedral 4-cell stages so as to enable quick identification of its sister. Preliminary trials in which this fluorochrome was used in conjunction with DiI showed that it too spread only to the sister of an injected blastomere. The fluorescein complexon-positive pair of blastomeres was then lysed with a sharp glass needle, and the resulting 2/4 conceptuses pooled separately for return to the oviduct according to whether they were the product of a meridional or an equatorial division. At recovery on the 8th day post-coitum, five morphologically normal gastrular stage conceptuses were obtained from fifteen 2/4 sister pairs resulting from equatorial division and six from eighteen 2/4 sister pairs resulting from meridional division.

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 animal–vegetal 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 1Go). 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, 1978Go). 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 8Go). 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 8Go). Specimens depicting this variability are shown in Figure 9Go.



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Figure 8. Diagrams illustrating a possible way of discriminating between the two products of meridional division (green) in regular tetrahedral 4-cell conceptuses when oriented with the three PB-adjacent blastomeres co-planar below the PB-remote product of the equatorial division (= red). Illustrated are three arrangements differing by 120° rotation about the animal–vegetal axis in which overlap of the PB-remote blastomere is greater (= asterisk) for the more clockwise (AC) versus less clockwise (DF) product of the meridional division.

 


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Figure 9. Examples of regular tetrahedral 4-cell conceptuses with the three blastomeres that are adjacent to the PB co-planar beneath the fourth one that is remote from this body. In each case the glass probe points to the sister of the blastomere that is remote from the PB, while the blastomere adajcent to the PB which overlaps the PB-remote one most extensively is marked with an asterisk. Thus, the blastomere that is remote from the polar body is overlapped most extensively by its sister in (A), by the more clockwise product of the meridional division in (B), and by the less clockwise product of this division in (C).

 
Cleavage planes in non-regular tetrahedral 4-cell conceptuses
The minority of 4-cell conceptuses that could not be classified as regular tetrahedrons varied considerably in form, typically having four to five intercellular contacts rather than six. Among these were specimens with an almost planar arrangement of blastomeres, in which the PB was either in contact with all four blastomeres (Figure 6A, BGo) or lay between just two (Figure 6C, DGo). Use of Dil-labelling to identify sisters yielded results that were consistent with the former being the product of meridional, and the latter of equatorial, division of both 1/2 blastomeres. In others, rather than being either meridional or equatorial, the division plane of one or both blastomeres was clearly intermediate at between 30 and 60° off the animal–vegetal axis. Such variability was evident both among conceptuses developing in vivo and in vitro.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The present study was undertaken to ascertain whether the regular tetrahedral form of the majority of 4-cell mouse conceptuses is due to meridional division of both 1/2 blastomeres, but with an accompanying ~90° rotation of the animal–vegetal axis of one of them, as has been claimed for the rabbit (Gulyas, 1975Go). This hypothesis was tested in two ways. The first was to determine how microspheres used to decorate the vegetal polar region of 1/2 blastomeres were distributed between the products of their division. Instead of being associated consistently with both daughter blastomeres, as expected, they were as often restricted to only one. This was usually the 1/4 blastomere that, according to the location of the PB, was remote from the animal pole. The second approach was to gelate the perivitelline space with the aim of inhibiting axial rotation of blastomeres in order to see if the formation of regular tetrahedral 4-cell conceptuses was thereby prevented. While the presence of the gel resulted in some alteration in overall form at the 4-cell stage, the proportion of conceptuses with one meridional and one equatorial cleavage plane matched the incidence of regular tetrahedrons among controls whose perivitelline space had not been gelated. However, the case that presence of the gel had prevented blastomere rotation depended on there being no major shift in the position of the PB with respect to its marker oil drop in the zona pellucida during second cleavage. An obvious concern was that the PB would not be expected to show such a shift if it were invariably attached to the non-rotating blastomere. This possibility could be discounted since the PB was commonly found to be tethered to the equatorially dividing 1/2 blastomere. Moreover, even without gelation of the perivitelline space, a marked shift in the position of the PB during second cleavage was significantly less frequent than its attachment to the equatorially dividing 1/2 blastomere.

According to Gulyas (1975)Go, 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 10GoGoGo). Consequently, 1/4 blastomeres can be assigned to three categories according to how much of the animal–vegetal 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 10Go). It should be noted that this differs from previous use of such blastomere notation (Gulyas, 1975Go; Graham and Deussen, 1978Go; Kelly et al., 1978Go) 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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Figure 10. (A) Diagrammatic representation of the regular tetrahedral 4-cell mouse conceptus showing the products of the meridional division, which are denoted the A/B pair, in green, and the PB-adjacent (C) and -remote (D) products of the equatorial division respectively in yellow versus red. D is readily identifiable in intact conceptuses because of its remoteness from the PB. Its sister, C, can only be differentiated reliably from members of the A/B pair via spread of a lineage label from D. Attempts to differentiate between members ofthe A/B pair have so far been unsuccessful. (B) Similar to A but rotated clockwise about the animal–vegetal axis so that one member of theA/B pair lies behind the other, i.e. the plane of first cleavage is roughly vertical. Note that D overlaps the vegetal end of the animal–vegetal axis instead of lying wholly to one side of it, thereby producing a bend in the originally straight first cleavage plane, as indicated by the dashed line.

 
On their own, A/B and C/D sister pairs gave comparable rates of normal development through gastrulation. However, since all levels of the animal–vegetal axis are represented in both types of sister pair, within individual blastomeres in one case and in separate ones in the other, this finding does not rule out the possibility that the three categories of 1/4 blastomere differ in developmental potential. Normal development to term has not been obtained from individual 1/4 blastomeres in the mouse, and the single recorded normal-looking early postimplantation conceptus appeared to be too retarded to develop much further (Rossant, 1976Go). However, in a very carefully controlled study (Somers et al., 1990Go), no difference was found in either the rate of implantation or of normal development thereafter between intact 4-cell conceptuses and those from which one blastomere had been removed. Assuming that there was no bias in the choice of the blastomere for removal, this finding makes it unlikely that the presence of either the C or D blastomere is indispensible for normal development. However, since it does not exclude indispensability of one of the A/B blastomeres, further investigation of the potency of specific 1/4 blastomeres from regular tetrahedral 4-cell conceptuses is clearly warranted.

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, 1989Go) 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 6Go). 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, 1978Go; Suzuki et al., 1995Go).

Recent studies suggest that in undisturbed development the equator of the blastocyst corresponds with the plane of first cleavage (Gardner, 2001Go; Piotrowska and Zernicka-Goetz, 2001Go). 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., 2001Go), the boundary between the clones was found to be rather variable and clearly not strictly orthogonal to the embryonic–abembryonic 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 10BGo). 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 animal–vegetal 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.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
I wish to thank Ann Yates, Tim Davies and Andy Forkner for help, Chris Graham for valuable discussion, and the Royal Society and the Wellcome Trust for support.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on June 19, 2002; accepted on August 8, 2002.