Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK
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Abstract |
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Key words: mouse blastocyst/mural trophectoderm/polarized growth/polar trophectoderm/trophectoderm
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Introduction |
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There is no direct evidence that the flow of cells from polar to mural trophectoderm is radial and, indeed, two recent findings raise the possibility that it is not. One is that the mouse conceptus is already bilaterally rather than radially symmetrical at the early blastocyst stage (Gardner, 1997), as was reported much earlier for the rat (Huber, 1915
). However, particularly since bilateral symmetry becomes temporarily obscured during blastocyst expansion (Gardner, 1997
), polar to mural flow of cells might nonetheless conform to a radial pattern. The other finding, which is more difficult to reconcile with a radially symmetrical flow of cells, emerged from a recent clonal analysis of growth of the polar trophectoderm (Gardner, 1996
). Here, ionophoretic injection of horseradish peroxidase was used to label either the central polar trophectoderm cell or a single peripheral one in 3.5 days post-coitum (d.p.c.) blastocysts which were then cultured for ~ 1 or 2 days thereafter. Clones formed by labelled central polar cells were typically displaced towards the abembryonic pole of the blastocyst, in accordance with the results of earlier studies (Cruz and Pedersen, 1985
; Dyce et al., 1987
). In contrast, only about one-quarter of the clones formed by labelled peripheral polar trophectoderm cells behaved thus, half retaining their ancestral location, and the remainder actually shifting towards, rather than away from, the centre of the polar trophectoderm during the first day of culture (Gardner, 1996
). Such variable deployment of peripheral clones suggests that the spread of cells from polar to mural trophectoderm is polarized rather than radially symmetrical.
In the present study the entire polar trophectoderm was labelled selectively with fluorescent latex microspheres (Fleming and George, 1987) so as to enable the overall pattern of flow of its cells into the mural region to be visualized. Regardless of whether they were cultured with or without the zona pellucida, or returned to the uterus for further development, labelled blastocysts almost invariably showed polarized rather than radially symmetrical spread of label into the mural trophectoderm thereafter. In additional blastocysts, individual junctional trophectoderm cells were labelled with a fluorescent tracer in order to determine whether they extended a process onto the blastocoelic surface of the ICM. Cells with such an extension showed no greater tendency to retain a junctional location than those without. Hence, the hypothesis that cells with an extension might thereby be held at the polarmural junction and thus circumferentially restrict egress of cells from the polar trophectoderm (Gardner, 1996
) appears untenable.
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Materials and methods |
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All blastocyst donor females on altered lighting were killed and their uterine horns flushed between 10:00 and 12:00 h on the fourth d.p.c., while those on standard lighting were killed and flushed between 14:00 and 15:00 h. MTF-HEPES (Gardner and Sakkas, 1993) was used as the medium for recovery, storage and manipulation of blastocysts, and for their transplantation to recipient uteri. Concepti that had not reached the early or expanding blastocyst stage (for staging, see Gardner, 1997) at the time of recovery were cultured until they did so. For growth in vitro, blastocysts were placed in pre-equilibrated drops of alpha medium (Stanners et al., 1971
) plus 10% inactivated fetal calf serum (
+IFCS) overlaid with light paraffin oil (BDH, Poole, UK) in 30 mm disposable tissue culture dishes, and incubated at 37°C in 5% CO2 in air.
Blastocyst manipulation
All manipulations were done using a Leitz micromanipulator assembly with the blastocysts immobilized by gentle suction on the tip of a holding pipette (Gardner, 1978; Gardner and Davies, 1998
). Global labelling of the polar trophectoderm was achieved by exposing it to fluorescent latex microspheres (Fluoresbrite YG, carboxylate; Polysciences, Warrington, PA, USA) of a size which its cells could readily endocytose. Several batches of microspheres whose mean diameter ranged from ~0.20 to 1.66 µm were used in different experiments. Three to four drops of a stock suspension in water were added to 25 ml of phosphate-buffered saline (PBS), MTF-HEPES, or
+IFCS which, depending on the size of the microspheres, was then passed through a sterile 0.22 or 0.45 µm filter. For batches of microspheres that were 0.5 µm or more in diameter, those that were retained were then resuspended by back-flushing up to 1.0 ml of medium through the filter from its distal end. For batches of ~ 0.20 µm, microspheres that passed through a 0.45 µm filter but were subsequently retained in a 0.22 µm filter were used. Immediately before being dispensed into culture or manipulation chamber drops, the final suspensions were agitated on a whirlimixer to disperse aggregates and then microfuged at ~2250 g for up to 30 s to sediment residual clumps.
Two different ways of exposing the polar trophectoderm selectively to the microspheres were adopted. One was to slit the zona pellucida over the polar region in order to obtain externalization of the ICM and overlying polar trophectoderm during subsequent culture so that only the latter would be exposed to the microspheres. The other was to inject a suspension of microspheres under the zona at the centre of the polar region.
Slitting of the zona was carried out as described previously (Tsunoda et al., 1986), taking care to ensure that the slit was both wide and well-centred on the polar trophectoderm. In later experiments, a pair of slits set approximately at right-angles to each other was made in the zona, rather than a single one. In most experiments, the polar trophectoderm was exposed briefly to a suspension of microspheres in Dulbecco A PBS once it had completed herniation in culture. Providing the blastocysts were first rinsed in PBS on removal from culture, the microspheres adhered rapidly to the exposed trophectoderm. In some later experiments, blastocysts were exposed to the microspheres during herniation by including them in culture medium. Following exposure of the polar trophectoderm to the microspheres, blastocysts were rinsed extensively before being examined briefly by fluorescence microscopy using standard fluorescein filters to check the quality of labelling (Figure 1
). They were then returned to culture directly or exposed briefly to acidified Tyrode's (AT) saline to remove the zona pellucida before further culture or transfer to the uteri of day 3 pseudopregnant females (Gardner and Davies, 1998
). No adverse effects on development have been discerned after single, or even repeated, exposure of preimplantation mouse concepti to 450490 nm light (Fleming and George, 1987
; Zernicka-Goetz et al., 1997
).
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To determine whether junctional trophectoderm cells had an extension onto the blastocoelic surface of the ICM they were labelled either ionophoretically with 10 kDa tetramethylrhodamine dextran-lysine-biotin (TMRDLB; Molecular Probes Inc., Eugene, OR, USA) in 0.1 mol/l KCl (Gardner and Cockroft, 1998), or by pressure injection with DiI (also from Molecular Probes) in mineral oil (Gardner, 1997
). Some blastocysts were examined in detail by fluorescence microscopy following injection: the remainder were examined just long enough to ascertain whether or not the labelled cell had an unequivocal extension before being cultured separately in vitro overnight for recording the distribution of the resulting clones during the following morning.
Scoring of labelled blastocysts
On recovery from culture or the uterus, blastocysts were returned to hanging drops in Puliv manipulation chambers for detailed examination by fluorescence microscopy and brightfield microscopy. A solid, fine-tipped siliconized glass needle was used to orient them appropriately, and a holding pipette to immobilize them for photography. Blastocysts that had been labelled with the strongly fluorescent microspheres were usually fixed first because they deteriorated very rapidly when examined fresh. For fixation, they were immersed for at least 40 min in 1% glutaraldehyde in PBS that included 10 kDa polyvinylpyrrolidone (PVP) at 4 mg/ml. Thereafter, the blastocysts were rinsed and examined either in deionized water containing PVP at 4 mg/ml or in MTF-HEPES diluted ~ 4-fold in deionized water so they remained expanded. The presence of the PVP prevented adhesion of the blastocysts to surfaces and their consequent damage.
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Results |
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With subzonal injection of the microspheres it was harder to control the distribution of labelling, so that a higher proportion of blastocysts had to be rejected. Commonly, the microspheres either became disseminated well beyond the limit of the polar trophectoderm or were confined to only part of it. This was because the perivitelline space often failed to open up uniformly as the suspension was ejected from the pipette, presumably because of local differences in the rigidity of trophectoderm cells or in the strength of their attachment to the zona.
The results of microsphere labelling of the polar trophectoderm by both methods are summarized in Table I. Not all recovered blastocysts were classified as scorable, for reasons that are given in the footnote to the table. Importantly, to guard against artefactual relocation of the ICM, all blastocysts which did not retain labelling throughout the polar trophectoderm were rejected. The highest proportion of unscorables, mostly due to damage to the polar trophectoderm, was in the herniation series where the zona was removed with AT saline prior to culture. Regardless of the method of labelling, or whether the zona was left on or removed thereafter, the great majority of scorable blastocysts in all series exhibited a single, circumferentially restricted, coherent patch of fluorescent cells in the mural trophectoderm (Table I
; Figure 2
). Outside this patch, the mural trophectoderm was either completely unlabelled or had the label confined to cells at its immediate junction with the polar trophectoderm. When blastocysts were viewed from the embryonic pole, the proportion of their circumference that the patch occupied was commonly around 50%, but ranged from ~ 25% to 70%. The distal limit of spread of the label also varied considerably, from proximal to the equator of the blastocyst to close to the abembryonic pole. Patches which extended further distally generally appeared narrower than those that penetrated the mural trophectoderm more modestly. More significantly, in the overwhelming majority of cases, the distal extremity of the patch lay diametrically opposite the centre of the region where the mural trophectoderm remained essentially unlabelled. Typically, spread of the label from the polar to the mural trophectoderm took the form depicted in Figure 3
. Most of the scorable blastocysts that did not exhibit this pattern showed a limited more or less radially symmetrical spread of label. That cases where spread was not obviously polarized were encountered, most commonly following subzonal injection of the beads, may be attributable to the greater difficulty in obtaining uniform labelling of the polar trophectoderm by this method. The remaining scorable blastocysts were particularly interesting in exhibiting two circumferentially limited patches of labelling in the mural trophectoderm that were diametrically opposite each other (Figure 4
). In one of these blastocysts the two patches extended about equally into the mural trophectoderm, while in all the others they did so unequally.
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In the non-cultured series, between one and eight individual junctional cells or cell pairs were labelled in each of 21 blastocysts, and a complete circumferential ring of eight such cells in a further blastocyst. Altogether, a little more than half the injected junctional cells or cell pairs (49/92) exhibited an unequivocal extension onto the blastocoelic surface of the ICM. While some extensions reached the centre, others obviously fell short of it. Regardless of length, the extensions varied in shape from broad triangles to much narrower, cable-like projections. In the blastocyst in which the complete circumferential ring of eight junctional trophectoderm cells was labelled successfully, three cells (an adjacent pair and a separate one) each had a long extension and a fourth a much shorter one, while the remaining four cells altogether lacked an extension. Examples of labelled junctional cells with an extension are shown in Figure 5.
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Discussion |
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Collectively, the above findings confirm the conclusion suggested by an earlier clonal analysis (Gardner, 1996) that the polar to mural flow of cells in the trophectoderm which accompanies growth of the blastocyst is normally polarized rather than, as had been assumed hitherto, radially symmetrical. That the flow was not discernibly polarized in all cases may be because, in order to assess the quality of labelling, all blastocysts had to be exposed to fluorescence microscopy before culture or transfer. Although both the duration and level of excitation were kept to a minimum, and blastocysts with obviously dead or excluded cells were discounted, the possibility remains that the polar trophectoderm occasionally suffered more subtle damage that perturbed its subsequent growth. The incidence of non-polarized spread was higher in blastocysts that were labelled by subzonal injection which also tended to give denser labelling. That radiation damage might also account for the several cases where spread of the label was bi- rather than uni-polar also cannot be excluded. It would, nevertheless, be intriguing to know whether cells egressed from opposite sides of the polar trophectoderm simultaneously or sequentially in these specimens.
The fact that the cells which leave the polar trophectoderm typically form a single coherent patch in the mural region argues that they must have emigrated in essentially the same direction throughout the 1520 h between labelling and scoring. What is not clear is whether they leave the polar region on a front that is as broad as the patch they eventually form in the mural trophectoderm, or whether they spread circumferentially after emerging more focally. Limited observations on blastocysts cultured for just a few hours after labelling suggest that spread of the label is not confined initially to a narrow segment of the polarmural junction (R.L. Gardner, unpublished data).
The question of how the spread of cells from polar to mural trophectoderm is restricted radially during blastocyst growth has been discussed elsewhere (Gardner, 1996). In particular, the possibility was considered that trophectoderm cells which extend a process over the blastocoelic surface of the ICM (Ducibella et al., 1975
; Fleming et al., 1984
) might thereby be anchored enduringly at the polarmural junction (Gardner, 1996
). It was argued that prevention of the net distal movement of junctional clones could restrict egress of cells from the polar region, provided that part of the junction was occupied by cells which were not so anchored. The results of lineage-labelling junctional cells reported here offer no support for this hypothesis. Thus, while not all junctional cells extend onto the ICM, those that do not tend to be interspersed among those that do. More significantly, clones formed by junctional cells with a extension showed net displacement murally during subsequent culture as often as those without one. Hence, extensions are clearly transient rather than enduring features of junctional cells and therefore cannot be responsible for the circumferential restriction in polar to mural flow during blastocyst growth. The incidence of junctional cells with an extension was found to be much lower than was expected from earlier findings (Fleming et al., 1984
). This may reflect better growth of blastocysts in vitro in the present study, since the proportion of junctional cells with an extension would be expected to vary inversely with the rate of polar to mural cell flow. Previous observations (Soltynska, 1985
) on freshly recovered blastocysts are consistent with this possibility. Whereas extensions were found to cover the surface of the ICM almost entirely in nascent blastocysts, in expanded blastocysts much of this surface was uncovered. Morphological observations consistent with withdrawal of extensions as cells are displaced murally are summarized diagrammatically in Figure 6
, which also indicates how such displacement might be achieved without disrupting the tight junctional permeability seal between adjacent trophectodermal cells through their partially overlapping each other.
One implication of the present findings is that distance from the ICM cannot be used as a measure of the length of time for which cells have resided in the mural trophectoderm. In view of the uniform way in which morphological giant transformation of mural cells spreads from the abembryonic pole, the present findings make it even less likely that this process bears any relationship to commitment to endoreduplication of the genome.
Finally, it is intriguing that whenever the label spread into the mural trophectoderm at two discrete sites rather than just one, these were always more or less diametrically opposite each other. It suggests that the orientation of flow of cells from the polar to the mural trophectoderm may be related to the axis of bilateral symmetry of the early blastocyst, a possibility that is now being explored.
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Acknowledgments |
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References |
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Submitted on June 8, 1999; accepted on November 16, 1999.