Mammalian Development Laboratory, University of Oxford, Department of Zoology, South Parks Road, Oxford OX1 3PS, UK
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Abstract |
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Key words: blastocyst axes/first cleavage plane/midpiece/mouse/sperm entry point
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Introduction |
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Rather than being generated intrinsically, the necessary asymmetry might depend on an extrinsic factor such as the fertilizing sperm. Particularly pertinent is a recent study in which the authors claim that the plane of first cleavage corresponds with the sperm entry point (SEP) in the mouse (Piotrowska and Zernicka-Goetz, 2001). Their consequent conclusion that the fertilizing sperm has a patterning role is based on the use of subzonal injection of Concanavalin A (ConA) and of phytohaemagglutinin (PHA)-treated fluorescent microspheres to achieve focal labelling of the surface of the zygote. However, the authors make no reference to past studies on binding of these lectins to early mouse conceptuses even though awareness of two findings is of crucial relevance to the approach they used. First, both lectins bind avidly to the zona pellucida (Nicolson et al., 1975
; Legge, 1991
) and, given its glycoprotein composition, probably also to the cortical granule envelope that occupies the perivitelline space (Hoodbhoy et al., 2001
). Second, unlike almost the entire remainder of the zygote, the fertilization cone that forms at the SEP does not discernibly bind ConA (Maro et al., 1984
). If this is due to the absence of microvilli (Shalgi et al., 1978
; Maro et al., 1984
), it may well also apply to PHA, in which case binding of PHA-treated microspheres to the SEP will differ in nature or strength from elsewhere on the zygote's surface.
In view of these earlier findings, there are clearly serious potential pitfalls in using lectins for focally labelling the surface of the zygote with the zona pellucida left on, as was done in the experiments of Piotrowska and Zernicka-Goetz (2001). Hence, validation of the use of PHA-treated microspheres as a surface marker depends crucially on demonstrating that they retain their relative position at least through the first cleavage division. The principal assay used by Piotrowska and Zernicka-Goetz failed to substantiate this because it entailed placing a microsphere either adjacent to, or diametrically opposite, the second polar body (PB). Given that first cleavage is usually approximately meridional, microspheres placed at either pole of the zygote should normally lie at or close to the cleavage plane. Hence, general movement of lectin-binding and other surface components towards the future cleavage plane, which has been reported both in various mammalian cells (Berlin et al., 1978; Koppel et al., 1982
; Wang et al., 1994
) and in sea urchin zygotes (McCaig and Robinson, 1982
), would go undetected. Showing that microspheres placed diametrically opposite each other in the equatorial region of zygotes prior to first cleavage remain opposite each other thereafter would seem to be necessary for determining whether they retain their relative position.
Thus, the study of Piotrowska and Zernicka-Goetz fails to provide sufficiently rigorous validation of the use of lectins for surface marking of the zygote to show, (i) that such marks retain their position, and (ii) do not influence the cleavage plane through mediating attachment between the zygote and the overlying zona pellucida.
Given the obvious importance of establishing whether the fertilizing sperm has a role in early patterning, the present study was undertaken to investigate the relationship between its components and the first cleavage plane using methods that avoid the uncertainties associated with surface labelling of the zygote. Particular emphasis was placed on investigating this relationship in conceptuses that were not subjected to any experimental manipulation and should therefore reveal the normal situation. We find the plane of first cleavage to be almost random with respect to the SEP, and are unable to detect binding of either ConA or PHA to the SEP of mechanically denuded living zygotes. Moreover, we find not only that both lectins tend to move towards the first cleavage plane, but that the coherent labelling for ConA that persists beyond the 2-cell stage is associated with the zona pellucida rather than the surface of the conceptus.
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Materials and methods |
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Inspection and manipulation of conceptuses
Zygotes, 2-cell conceptuses and blastocysts were inspected and micromanipulated in individual hanging drops in a Puliv chamber filled with heavy paraffin oil using differential interference contrast (DIC) or epifluorescence (FM) microscopy with either a x40 or a x60 water immersion objective lens. For recording the location of the anterior end of the sperm tail (AnT) without intervention in early zygotes, its site was first marked with an oil drop in the overlying zona pellucida, as described elsewhere (Gardner, 2001). Zygotes were then examined with their AV axis horizontal so as to enable the proportionate distance of the oil drop from the animal pole to be estimated. Thereafter, they were reoriented with the AV axis vertical and the approximate angle of the oil drop relative to an axis defined by the centres of the first and second PBs, or by the second PB and its midbody was noted (Figure 1AC
). Following this, each zygote was cultured separately for re-examination either during first cleavage or at the early 2-cell stage to see if the orientation of axes defined by the PB(s) appeared to have been conserved. Where conservation of one or both axes was indicated, a small oil drop was injected cortically according to the axial and circumferential coordinates recorded for the AnT at the early zygote stage.
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The solution used to permeabilize zygotes and 2-cell stages was as described (Schatten et al., 1989), except that the concentrations of EDTA and Triton-X were raised to 1 mmol/l and 3% respectively. Initially, permeabilization was for several hours, though later in the study 1 min at room temperature was found to suffice. Thereafter, the conceptuses were rinsed briefly in an aliquot of permeabilization solution from which Triton-X and glycerol had been omitted before being transferred to a further aliquot of this rinse containing 5 nmol/l OGPT prepared in dimethylsulphoxide. After incubation in this solution for 10 min in the dark, conceptuses were transferred via a further rinse to drops of KSOMHEPES in a Puliv chamber for scoring by microscopy.
Marking of the AnT with oil drops in the zona pellucida
Marking the location of surface features of zygotes by injecting oil drops into the zona pellucida was done as described elsewhere (Gardner, 2001). To prevent their often considerable rotation within the zona pellucida, zygotes were first incubated for 1.251.5 h in KSOM containing 1.1% (w/v) low viscosity sodium alginate (Sigma, Poole, Dorset, UK). After a brief rinse in KSOMHEPES, they were exposed for up to 40 min at room temperature to a solution of 1.5% CaCl2·2H2O and 0.90% NaCl in analar water that had been diluted 10-fold with KSOMHEPES so as to induce slow gelation of the alginate within the perivitelline space. Thereafter the AnT, the second PB and, where present, the first PB were marked distinctively by injecting, respectively, one small, two large, and one large oil drops into the zona pellucida or, in some cases, into the gelated perivitelline space. All zygotes were then cultured in KSOM minus alginate for harvesting during or shortly after first cleavage. Finally, they were returned to a Puliv chamber for checking from the relationship between the PB(s) and their marker oil drops whether they had rotated in the zona pellucida. Those showing no rotation had an oil drop injected into the periphery of the vitellus in alignment with the AnT marker oil drop. To prevent wrinkling during subsequent permeabilization, such conceptuses were first incubated for 15 min at room temperature in phosphate-buffered saline (Dulbecco A, Oxoid, Basingstoke, Hants, UK) supplemented with 4 mg/ml of 10 kDa polyvinylpyrrolidone (PVP) and containing 1 mmol/l EDTA to dissolve the gel in the perivitelline space. They were then permeabilized and treated with OGPT, as described earlier, before being examined microscopically.
Labelling of sperm
Sperm recovered from both vasa deferentia of PO male mice were incubated at 37°C for 10 min in 102 µl of Ca2+/Mg2+-free Tyrode's saline with 10% KSOM under light paraffin oil to allow them to become well dispersed. For labelling the mitochondrial sheath that invests the midpiece (Bishop and Walton, 1960; Fawcett, 1981
), 50 µl of 1 µg/ml MitoTracker Green (MT Green, M-7514; Molecular Probes Inc.) in Tyrode's/KSOM was added to the suspension at a final concentration of 490 nmol/l, and the sperm were then incubated for a further 610 min. In some experiments, tetramethylrhodamine isothiocyanate (Sigma, T3163) was also included in this solution at a final concentration of 0.261.3 mmol/l. The labelled sperm suspension was then used for artificially inseminating female mice shortly after the end of the dark period during which they had mated with vasectomized males (Kile, 1951
). The females were killed ~1 day after artificial insemination (AI) and all morphologically normal-looking 2-cell conceptuses were examined by FM. Where specific focal fluorescence was observed following labelling of sperm with MT Green alone, its location was marked in some conceptuses by injecting a small oil drop in the vitellus. These conceptuses were then permeabilized and stained for identifying the AnT. In the remaining conceptuses, the outer surface of the blastomere containing the focus was marked with a pair of oil drops in the zona pellucida before they were cultured to the blastocyst stage when the position of the marker oil drops along the EmAb axis was recorded. All 2-cell conceptuses obtained by AI using sperm that had been double-labelled with both MT Green and tetramethylrhodamine isothiocyanate were examined by FM to compare the distribution of red and green signals in the vitellus.
For mapping the location of the AnT in permeabilized first cleavage and 2-cell stages, its location was first marked with an oil drop so that measurements could be made on the screen of a monitor that was coupled to a video camera mounted on the microscope. Each conceptus was first oriented with its AV axis horizontal. Both the distance of the AnT marker oil from the animal pole and the total length of the AV axis were then measured and the percentage distance of the AnT along the axis calculated (Figure 1D). The conceptus was then oriented with its animal pole uppermost and its actual or, in the case of first mitotic stages, its putative or nascent, cleavage plane vertical. Finally, the angle of departure of the AnT marker oil from the centre of the cleavage plane was measured with a protractor (Figure 1E
). To facilitate comparison of these findings with those of Piotrowska and Zernicka-Goetz (2001), the mean values of the angles embracing the inner and the central thirds of blastomeres were determined from measurements on both 2-cell PO and F1xF1 conceptuses (Figure 1F
).
Lectin binding
For investigating binding of fluorochrome-conjugated ConA and PHA to the fertilization cone, living early zygotes were denuded mechanically using two fine-tipped glass needles held on micromanipulators (Leitz, Luton, Bedfordshire, UK). This was done to avoid the risk of altering the surface of the zygote by exposing it to acidified Tyrode's saline or the proteolytic enzymes that are normally used to eliminate the zona pellucida. Once denuded, zygotes were rinsed briefly in phosphate-buffered saline (PBS) before being exposed to ConA (Alexa-Fluor ConA; Molecular Probes, Inc.) at 500 or 20 µg/ml or to rhodamine isothiocyanate-conjugated PHA (Sigma, Poole, Dorset, UK) at 300 or 20 µg/ml. The surface of additional zygotes was labelled focally by injecting a small volume of 500 µg/ml ConA against it via a micropipette inserted through the zona pellucida, as described elsewhere (Piotrowska and Zernicka-Goetz, 2001). Zygotes treated thus were then cultured to the blastocyst stage.
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Results |
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According to this assay, the AnT remained at its initial location in the majority of cases, and seldom lay far from it (Table I). Importantly, even when it had remained at its original location it showed no greater tendency to lie closer to the cleavage plane than when it had not. Thus, it was remote from the cleavage plane in 5/8 2-cell conceptuses, 2/2 anaphases, and 1/1 metaphases in which it lay near the equator and had not shifted circumferentially.
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In 12 of 15 2-cell conceptuses obtained from AI with double-labelled sperm in which red fluorescence due to tetramethylrhodamine isothiocyanate was detected, it co-localized very precisely with the MT Green signal (Figure 7DI). Each of the remaining three conceptuses also had an additional small focus that exhibited only red fluorescence. This focus was near the cleavage plane in two cases and remote from it in the third.
Binding of ConA and PHA to the zygote
All findings reported here were based on examination of at least 12 specimens. Before being exposed to either lectin, early zygotes from matings between PO or F1 mice were denuded of the zona pellucida mechanically with micro-needles so as to avoid chemical modification of their surface by the reagents that are normally used to remove this envelope. Relative to the remainder of the surface of the zygote, the fertilization cone showed essentially background fluorescence with both ConA and PHA (Figure 8A, B and E, F), even when exposed to the very high concentration of the lectins used by Piotrowska and Zernicka-Goetz (300500 µg/ml) at which non-specific binding may occur (Konwinski et al., 1977
). When the isolated zonae pellucidae were exposed to the lectins, they fluoresced very intensely throughout (Figure 8C, D and G, H
). When part of the vitelline surface of early zygotes was labelled by restricted subzonal injection of ConA this sometimes led to such strong adherence between the surface of the vitellus and the zona pellucida as to cause the zygote to lyse during subsequent mechanical exzonation. In zygotes that were cultured within an intact zona pellucida, much of the ConA or PHA was endocytosed by the early 2-cell stage and, regardless of the original site of labelling, what remained on the surface tended to be concentrated towards the cleavage plane (Figure 8I, J
). All zygotes focally labelled with ConA that were cultured to the blastocyst stage showed a persisting coherent patch of fluorescence. However, following mechanical denudation or spontaneous hatching of such blastocysts, the patch was invariably found to be associated with the zona pellucida. This was obvious even without recourse to removing the zona pellucida if labelled conceptuses were rotated during examination at any stage before blastocyst expansion when the perivitelline space disappears (Figure 8K
). In no case was coherent fluorescence found to persist beyond the 2-cell stage at the surface of the conceptus itself.
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Discussion |
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Hitherto, the distribution of components of the fertilizing sperm have not been investigated systematically either during or after first cleavage in the mouse. This is presumably because they are harder to discern in this species than in the rat (Blandau and Odor, 1952). Nevertheless, use of permeabilization in conjunction with OGPT staining gave clear enough resolution of the tail to allow its anterior extremity to be distinguished from its progressively unravelling posterior region in both late zygotes and 2-cell conceptuses. Moreover, AI with MT Green-labelled sperm enabled localization of the mitochondrial sheath which surrounds the anterior part of the tail in intact sperm (Fawcett, 1981
). In both PO and F1xF1 early 2-cell stages the AnT was more often remote from, than near to, the plane of first cleavage, regardless of its position along the AV axis (Figure 4
). This was also true for additional series of PO conceptuses examined during first cleavage (Figure 5
), both at metaphase when the plane of cytokinesis may not yet have been specified in all cases, and during anaphase when it clearly must have been (Rappaport, 1974
). Furthermore, both the direction in which the remainder of the tail was oriented and its overall shape were highly variable, effectively discounting the possibility that any part of it bore a consistent relationship to the plane of first cleavage.
Most importantly, where the AnT was superficial enough for its position to be mapped strictly non-invasively at the early zygote stage and this site could later be marked confidently with oil, it was found in the majority of cases to have remained at its original location through to, and even beyond, first cleavage. Since the AnT lies initially at the SEP (Figure 2), the latter must normally bear the same relationship to the plane of first cleavage as the former. That instances of obvious disparity between the AnT and its marker oil occurred following gelation of the perivitelline space argues that these were due, at least partly, to occasional shift in the position of the AnT rather than the PB(s) used to map its location. Such shifts might depend on proximity of parts of the tail to microtubule organizing centres orchestrating pronuclear migration.
In each of 17 early 2-cell conceptuses in which the main focus of the MT Green signal was marked with an oil drop, the drop was found to be close to the anterior region of the sperm tail. Moreover, where the mitochondrial sheath was identifiable directly by DIC, it invariably lay by or over the anterior region of the tail (e.g. Figure 3A, inset). In view of its consistent co-localization with the AnT, separate mapping of the relationship of the midpiece to the plane of first cleavage was considered unnecessary. Parenthetically, the above findings do not accord with an early claim that the midpiece mitochondria become dispersed throughout the cytoplasm of the mouse zygote before it reaches an advanced pronuclear stage (Gresson, 1940
).
Where a vitelline focus of red fluorescence was seen in 2-cell conceptuses following AI with double-labelled sperm, it almost invariably co-localized precisely with the MT Green fluorescence. Hence, the mitochondrial sheath seems the most likely candidate for the superficial patch of fluorescence in zygotes and cleavage stages from mice that had been artificially inseminated with fluorescein isothiocyanate- or tetramethylrhodamine isothiocyanate-labelled sperm (Gabel et al., 1979).
Apart from the mitochondrial sheath and the remainder of the tail, another internal component of the mouse sperm is the perforatorium which, being adjacent to the nucleus, must also lie initially at the SEP. This structure proved impossible to identify unequivocally either by DIC or FM, although Cummins et al. claimed to be able to distinguish it by FM following direct microinjection of MT Green-labelled sperm or their components into the cytoplasm of oocytes (Cummins et al., 1997). If, despite our failure to recognize it in early 2-cell stages after AI with MT Green-labelled sperm, it was nonetheless labelled sufficiently to contribute to the observed fluorescent foci, then it too must invariably have been associated with the anterior end of the sperm tail. In addition, the protein(s) equatorin, which is associated with a ladder-like structure in the pouch formed in the equatorial region of the sperm head between the persisting parts of the outer and inner acrosomal membranes (Manandhar and Toshimori, 2001
), has also been found to enter the oocyte on sperm penetration. While initially adjacent to the developing pronucleus, it later becomes separated from it and is typically associated with one blastomere at the 2-cell stage, but is said to be undetectable thereafter. Although the location of the residual equatorin-positive body relative to the cleavage plane has not been mapped systematically, it is remote from this plane in the 2-cell conceptus in which it is illustrated (see Manandhar and Toshimori, 2001
, Figure 3E
).
Hence, none of the components of the sperm that enter the oocyte has been found to bear a sufficiently consistent relationship to the plane of first cleavage to be instrumental in its specification. A further possibility to consider is whether, following its fusion with the oocyte, part of the surface membrane of the fertilizing sperm might continue to mark the site of sperm entry in an enduring way. This would run counter to general experience with membrane fusion where complete mixing seems inevitably to be the eventual outcome. From available data, this also appears to be true for fertilization (Gaunt, 1983). Possibly because it is organized maternally rather than paternally, the first mitotic spindle is unusual in being barrel-shaped and anastral in the mouse (Schatten et al., 1989
). It is therefore conceivable that its mode of orientation and, in consequence, the process of specification of the plane of first cleavage, could differ from that in other species. There is, however, no reason to suppose this might be the case. Nevertheless, according to the results of non-invasive mapping, the AnT often continues to mark the SEP despite its very variable relationship to the first cleavage plane.
Overall, when mapping of the AnT, and by association its midpiece and possibly the perforatorium, relative to the cleavage plane is compared with that of the PHA-treated microspheres that Piotrowska and Zernicka-Goetz (2001) used to mark the SEP, a striking disparity is evident. The microspheres most often mapped close to the cleavage plane during first cleavage and to the inner third of the positive blastomere thereafter. In contrast, the AnT was in the majority of cases remote from this plane both during and after first cleavage (Figures 4 and 5). A tendency for the SEP to be orthogonal to the cleavage plane rather than aligned with it could be explained if the long axis of the first mitotic spindle lies in the plane in which the two pronuclei come together. Regardless, it is clear that the present findings offer no support for the notion that the plane of first cleavage is related to the SEP in the mouse. For reasons enumerated earlier, we suggest that Piotrowska and Zernicka-Goetz (2001) have been misled through the use of lectins as markers. The present study has not only confirmed earlier findings that ConA does not bind detectably to the SEP and does so avidly to the zona pellucida (Maro et al., 1984
; Legge, 1991
), but has revealed that this is also true for PHA. This, in conjunction with evidence from both somatic cells and zygotes of movement of bound lectins towards the cleavage plane (Berlin et al ., 1978
; Koppel et al., 1982
; McCaig and Robinson, 1982
; Wang et al., 1994
), suggest that the association between the first cleavage plane and SEP reported by Piotrowska and Zernicka-Goetz (2001) is artefactual.
A further implication of the study by Piotrowska and Zernicka-Goetz (2001) is that following surface labelling of the zygote, ConA persists as a coherent patch on the surface of the conceptus through to the blastocyst. If true, this would provide a very valuable way of fate-mapping between the zygote and blastocyst stage. However, no evidence was provided that the patch persists on the conceptus rather than in the overlying zona pellucida, which is necessary in view of the marked propensity of blastomeres to endocytose this lectin (Handyside, 1980). Our own findings reported here have revealed that enduring coherent ConA labelling is entirely in the zona pellucida, and that little of the lectin associated with the vitellus remains on its surface beyond the first cleavage. For the purposes of their experiments it would not matter if the ConA patch were in the zona pellucida, providing the conceptus did not rotate within it. However, while the conceptus usually undergoes little or no rotation within the zona pellucida from the 2-cell to blastocyst stage (Gardner, 2001
), this is clearly not true before first cleavage (T.J.Davies and R.L.Gardner, unpublished observations).
Although the present findings offer no support for the claim that the fertilizing sperm is instrumental in specifying the plane of first cleavage, they do not discount the possibility that it might play other roles in early patterning. Gabel et al. did not follow up their observations to ascertain whether the fate of the blastomere with the patch of fluorescence differed consistently from that of its sister (Gabel et al., 1979). Here, it was found that the blastomere with the MT Green fluorescence tended to contribute more often to the embryonic than the abembryonic hemisphere of the blastocyst. However, the bias was much less striking than reported in two recent studies implying a link between precocious division and embryonic polar fate of the blastomere inheriting the SEP. In the first, in which a PHA-treated microsphere was placed at the SEP, the microsphere was found on the first blastomere to divide in 75% of 3-cell conceptuses (Piotrowska and Zernicka-Goetz, 2001
). In the second study, the earlier dividing blastomere was found to contribute predominantly to the embryonic hemisphere in >80% of blastocysts (Piotrowska et al., 2001
). It should be noted, however, that the interval between division of the two blastomeres varies greatly, so there is a risk of bias in limiting observations to specimens with an enduring 3-cell stage (Kelly et al., 1978
). Various differences between the two blastomeres of 2-cell conceptuses have been documented in the mouse. For example, it has been claimed that 2-cell blastomeres are unequal in size and that the larger of the two is the first to divide (Lewis and Wright, 1935
), but this has not been substantiated by subsequent work. Differences in dry mass occur early in the second cycle and the disparity increases as it progresses (Abramczuk and Sawicki, 1974
). Sister 2-cell blastomeres seem also to differ in the time at which nucleoli acquire staining properties thought to be indicative of onset of rRNA synthesis (Engel et al., 1977
), and in the duration of DNA synthesis (Luthardt and Donahue, 1975
).
Apart from the microvillus-free region immediately overlying the second meiotic spindle (Johnson et al., 1975; Eager et al., 1976
), there seem to be no obvious differences axially in the likelihood of attachment of the fertilizing sperm to the oocyte in the mouse. However, in the absence of discernible radial asymmetry in the living mouse oocyte, whether the site of sperm attachment is completely unrestricted orthogonally to the AV axis is uncertain. Hence, the possibility has yet to be excluded that both the site of sperm entry and the plane of first cleavage depend on organization of the oocyte.
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Acknowledgements |
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Notes |
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Plusa and colleagues (Plusa et al., 2002) claim that PHA-treated fluorescent microspheres behave similarly when used to label denuded as well as zona-intact zygotes. However, neither in these experiments, nor in another in which IVF with Alexa Fluor-labelled sperm was used to mark the sperm entry point, is evidence presented that either marker retains its ancestral position through first cleavage.
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References |
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Submitted on February 21, 2002; accepted on April 29, 2002.