cell membrane regions in preimplantation mouse …cell membrane regions in preimplantation mouse...

/ . Embryol. exp. Morph. Vol. 59, pp. 89-102, 1980 8 9Printed in Great Britain © Company of Biologists Limited 1980

Cell membrane regions in preimplantationmouse embryos

By L. IZQUIERDO1, T. LOPEZ1 AND P. MARTICORENA1

From the Department of Biology, Faculty of Science,University of Chile

SUMMARY

Cell membrane regions characterized by alkaline phosphatase activity are described incleaving mouse embryos and early blastocysts. Enzyme activity is demonstrated by light andelectron microscopy, from the late 4-cell stage onwards, on the cell surfaces between blasto-meres but not on the outer surface of the embryo. Experiments with dissociated morulaeshow that this is probably not an artifact due to the retention of the enzyme reaction productbetween the blastomeres. With the electron microscope the activity is also demonstrated incrystalloid bodies within the cytoplasm. The localization and growth during cleavage of cellmembrane regions with enzyme activity is interpreted as the result of new cell membraneformation and/or as a relation of the crystalloid bodies with the cell membrane through thecortical system of microtubules and filaments.

INTRODUCTION

Since the first demonstration by Mulnard (1955) of non-specific alkalinephosphatase activity (APA) in early mammalian embryos, several researchgroups have been attracted to the subject because the onset of the enzymeactivity and its localization suggest a pattern of cell differentiation and morpho-genesis (Dalcq, 1957; Sherman, 1972; Solter, Damjanov & Skreb, 1973;Mulnard, 1974; Izquierdo & Marticorena, 1975; lzquierdo & Ortiz, 1975;Izquierdo, 1977; Vorbrodt, Konwinski, Solter & Koprowski, 1977; Johnson,Calarco & Siebert, 1977; Mulnard & Huygens, 1978).

Regarding the onset of APA, the information was controversial until a directbiochemical assay showed the sudden appearance of the enzyme activity in8-cell mouse embryos. For further precision, individual embryos were testedby means of a cytochemical method and the enzyme activity was demonstratedin some late 4-cell embryos (Izquierdo & Marticorena, 1975). Observationson mouse, rat and hamster embryos, as well as on mouse embryos culturedin vitro, confirmed and extended our previous cytochemical results (Ishiyama& Izquierdo, 1977).

As far as the localization of APA in morulae and blastocysts is concerned1 Authors' address: Department of Biology, Faculty of Science, University of Chile,

Casilla 653, Santiago, Chile.

90 L. IZQUIERDO, T. LOPEZ AND P. MARTICORENA

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Cell membrane regions in preimplantation mouse embryos 91reports are at variance on whether inner and outer cells stain differentially.These contradictory observations have been ascribed to artifacts caused eitherby unreliable cytochemical methods or by the unequal thickenss of the embryos,when examined in whole mounts (Wilson, Bolton & Cuttler, 1972; Solter et ah1973). However, recent reports on APA localization are largely in agreement.Electron microscope observations of mouse morulae and blastocyst stained byvarious modifications of the Gomori-Takamatsu method show that APA isconfined to the cell surface between blastomeres and to crystalloid bodiesfound in the cytoplasm, and is detected neither at the outer surface of theembryo nor at the lining of the blastocoel (Izquierdo, 1977; Mulnard & Huy-gens, 1978). Light microscope observations on mouse embryos stained by anazo-dye cytochemical method show a somewhat similar distribution of APA(Johnson et al. 1977). The disparate results of Vorbrodt et al. (1977) havebeen attributed to unspecific staining produced by long incubation at a relativelylow pH (Mulnard & Huygens, 1978).

In this paper we deal with the possibility of an artifact due to the retention ofthe enzyme reaction product between the blastomeres, then we describe theregionalization of the cell membrane as revealed by APA and finally we discussthe mechanisms that might be involved. Our results support the idea that during

FIGURES 1-8

Fig. 1. Whole mount of an early 4-cell stage (42 h of development). No APA isrecognized.Fig. 2. Whole mount of an 8-cell stage (57 h of development). APA is observedbetween blastomeres.Fig. 3. Whole mount of two morulae of about 10 cells (60 h of development). Embryoat the left side reveals APA, embryo at the right side is a control incubated withoutsubstrate.Fig. 4. Whole mount of a pair of blastomeres isolated from a morula of about 10cells (60 h of development) and immediately processed for APA demonstration. APApatches are observed between blastomeres and on the surfaces exposed by dissocia-tion (arrows).Figs. 5 and 6. A whole mount of one pair of blastomeres isolated from a morula ofabout 12 blastomeres (61 h of development) photographed in two positions. Aftertheir isolation from the morula the pair was cultured in vitro for 17 h before fixationand APA demonstration. An APA patch is shown between the blastomeres, but noother patches are observed on the surface.Fig. 7. Whole mount of a morula of about 16 cells (64 h of development), halved intwo parts and cultured in vitro for 15 h before fixation and APA demonstration.The part represented shows APA between the compacted blastomeres and a patchon the surface exposed by halving (arrows).Fig. 8. Whole mount of a 2-cell stage (38 h of development) fused to a morula ofabout 16 cells (62 h of development) and cultured in vitro for 5 h before fixation andAPA demonstration. No APA is observed in the 2-cell stage nor in its contact withthe morula. The morula shows the typical localization of APA at this stage.

92 L. IZQUIERDO, T. L6PEZ, AND P. MARTICORENAT'

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Cell membrane regions in preimplantation mouse embryos 93

cleavage an intrinsic spatial pattern gradually unfolds, providing cells withinformation about their position in the embryo (Izquierdo, 1977).

MATERIALS AND METHODS

Preimplantation embryos were obtained from natural matings of Swiss-Rockefeller mice. Their stage is indicated in number of cells or in hours ofdevelopment, reckoned since the beginning of the day when the vaginal plug isfound.

The embryos recovered from the oviduct were either immediately processedfor APA demonstration or they were first treated so as to dissociate blastomeresor associate embryos. To this end, eggs were denuded of the zona pellucidaby pipetting them in 5 % Pronase (Calbiochem) in Biggers's culture medium(Biggers, Whitten & Whittingham, 1971) devoid of albumin, for 4 min at roomtemperature; the embryos were then rinsed in Biggers's medium and manipu-lated under mineral oil with fluid jets produced by a pipette and with the helpof gentle touches. Thereafter they were either fixed, or cultured in vitro beforefixation. Fixation lasted for 60 min in cold 3 % glutaraldehyde in 0-05 Mcacodylate buffer at pH 7-4. After fixation, embryos were rinsed in buffer for30 min and incubated for 90 min at room temperature in an alkaline leadcitrate medium at pH 9-5 (Mayahara, Hirano, Saito & Ogawa, 1967). Bestresults were obtained when the medium was buffered with tris-maleic acid andstabilized with 8 % glucose (Sundstrom & Mornstadt, 1975).

For light microscopy, after incubation the embryos were rinsed in buffer andthe enzyme reaction product was developed with ammonium sulphide. Theembryos were then immersed in glycerol and whole mounts observed underoblique illumination so as to cast shadows (Hlinka & Sanders, 1970) whichclearly reveal the contours of the nuclei and blastomeres, though this illumina-tion somewhat hinders an accurate recognition of APA localization on blackand white photographs.

For electron microscopy, embryos were rinsed in buffer (pH 7-4) and post-fixed for 60 min in 1 % OsO4 in cacodylate buffer; then they were dehydrated inacetone and embedded in low-viscosity resin (Spurr, Polyscience). Thin sections

FIGURES 9—14

Figs. 9-11. Morulae of 8 cells (60 h of development). The reaction product accumu-lates on the outer surface of the plasma membrane at the edge of the cleavagefurrows.Figs. 12 and 13. Morula of 8 cells (60 h of development). Discrete patches of APAon the surface of adjoining cells. Fig. 12 shows that the perivitelline space (pvs) iscontinuous with the interblastomeric cleft.Fig. 14. Morula of about 16 cells (65 h of development). Extended APA patches onthe cell membrane between blastomeres. APA is also detected in crystalloid bodies(cb) within the cytoplasm.7 EMB 59


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were stained with uranyl acetate and observed in a Philips EM 300 electronmicroscope.

Controls were performed on embryos whose enzyme was heat-denaturatedafter fixation and on embryos which were incubated in a substrate-free medium,keeping all other steps of the procedure unchanged. Out of a total number ofapproximately 300 embryos studied, 45 were observed with the electron micro-scope.

RESULTS

Light microscopy of whole embryos and electron microscopy of thin sectionsconfirm previous observations in that no APA is detected prior to the late 4-cellstage (see Introduction). The results concerning the localization of APA inlater stages of development will be summarized in this order: light microscopeobservations on intact embryos and on manipulated embryos, and observationswith the electron microscope.

Light microscopy. From the late 4-cell stage onwards (Fig. 1) while APA isneatly observed on the cell surface between blastomeres, no activity is detectedon the outer surface of the embryos nor is it possible to affirm that there isactivity on cytoplasmic structure (Figs. 2, 3). If embryos are heated afterfixation or incubated in a substrate-free medium, no traces of APA are detected(Fig. 3).

When pairs of blastomeres are dissociated from a morula, processed for APAdemonstration and observed in whole mounts, interblastomeric patches areevident and APA patches can also be recognized on the cell surface that hasbecome outer surface by virtue of the dissociation procedure (Fig. 4). If thedissociated pairs are cultured overnight before APA demonstration, the outerpatches are not always evident (Figs. 5, 6); actually, they are more clearly seenif morulae were dissociated into portions with several blastomeres each (Fig. 7).The variability of these results and the limited number of observations do notallow a systematic description, but they support the reality of outer APA patcheson the cell surface exposed by dissociation.

F I G U R E S 15-19

Fig. 15. Morula of about 16 cells (65 h of development). Discrete APA patches obser-ved on the cell surface between three blastomeres.Fig. 16. Morula of about 16 cells (65 h of development). Typical crystalloid bodiesin the cytoplasm showing APA.Fig. 17. Advanced morula (80 h of development). A wide interblastomeric spaceshows APA on both cell surfaces.Fig. 18. Advanced morula (80 h of development). The nucleolus (nu) and cytoplas-mic organelles do not show APA. The activity is found on the cell surfaces.Fig. 19. Advanced morula (80 h of development). In a wide interblastomeric space(arrow) no APA is shown on the cell membranes facing each other.

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Figs. 20 and 21. Advanced morulae (80 h of development). APA is detected onthe surface of blastomeres and in crystalloid bodies (cb). No APA is detectedin the nucleolus (nu) nor in mitochondria nor other cytoplasmic organelles, exceptcrystalloid bodies.


By fusing synchronous or asynchronous morulae in pairs we tried to converta part of their outer surface, which never reveals APA, into interblastomericsurface, which always does. After 5 h in culture the fused embryos were processedfor APA demonstration and observed in whole mounts. Under these conditionsthe artificial interblastomeric surfaces do not reveal any APA (Fig. 8).

Electron microscopy. In early morulae, APA is localized on the cell membraneboth at the advancing edge of the cleavage furrows (Figs. 9-11) and in discretepatches on adjoining blastomers (Figs. 12, 13). In late morulae and blastocysts,APA patches have spread out, and comprise most of the cell membrane border-ing the interblastomeric clefts (Figs. 18, 20-25).

Although the cytochemical procedure interferes with a clear definition of thecell membrane, the enzyme reaction product appears to be lumped on the outersurface while the inner surface shows a smooth profile. Narrow interblastomericspaces usually appear full of lead phosphate, but when the spaces are sufficientlywide, the reaction product may be detected on both cell membranes or onneither of them, as if cell membranes facing each other were symmetricalregarding their APA localization (Figs. 17-19).

In all developmental stages studied, the cell membranes around the embryoappear to be devoid of APA. The transition from the outer inactive region to theactive region between blastomeres is quite abrupt, even in early morulae whenapical tight junctions are still absent (Fig. 12). The cell surface bordering theblastocoelic cavity lacks APA, and as a consequence APA patches are exhibitedexclusively between trophectoderm cells (Fig. 23). Lead phosphate grains areusually found scattered in the blastocoel associated with cellular debris whichdo not reveal clear-cut membrane structures.

The membrane of the many diverse cytoplasmic organelles does not showAPA, and this is also the case for non-membranous structures except forcrystalloid bodies, which are known to arise during cleavage in mammalianembryos of several species (see Calarco & Brown, 1969; Van Blerkom, Manes &Daniel, 1973). In the mouse embryo, crystalloid bodies appear as structures ofapproximately 0-5 /«n in diameter that reveal a substructure periodicity of120-130 nm in the tightly packed crystalline form. These structures may also berecognized in diverse transitional forms, from typical crystalloid bodies to anetwork of loosely associated cross-striated microtubules or filaments.

Crystalloid bodies may show a strong APA (Figs. 14, 16, 21) or no activityat all (Fig. 23), even at the same stage of development; the less structured formsdo not show APA. We have not observed an association of crystalloid bodieswith the APA patches nor with any other part of the cell membrane.


pvs

24


DISCUSSION

Mulnard & Huygens (1978) analysed several possible causes of false localiza-tion of APA in preimplantation embryos but they did not consider an eventualartifact caused by the retention of the enzyme reaction product in the narrowclefts between blastomeres while it freely diffuses out from the surface aroundthe embryo and from the surface bordering the blastocoel. In fact, lead phos-phate is insoluble in our experimental conditions; however, this artifact wouldclosely mimic the APA distribution we describe in intact embryos. The presenceof APA patches on the new outer surface produced by dissociating morulae andthe absence of APA patches on the new interblastomeric surface produced byfusing embryos is inconsistent with an artifact due to differential retention oflead phosphate, though further investigation may be required to dismiss itentirely. On these bases we submit that the pattern of APA distribution inpreimplantation embryos reveals a true regionalization of the cell membranethat deserves to be discussed.

Considering first, that a large supply of cell membrane is needed during clea-vage; second, that APA is detected on the cell membrane (Izquierdo, 1977;Mulnard & Huygens, 1978; this paper); third, that the activity only arises atthe late 4-cell stage (Izquierdo & Marticorena, 1975; Ishiyama1 &} Izquierdo,1977) one would expect to recognize 'new' cell membrane if it] is] added indiscrete lots to the cell surface (Izquierdo, 1977). Indeed, our observationssuggest that 'new' membrane is formed at the leading edge of the cleavagefurrow, leaving symmetrical patches between blastomeres when their divisionis completed. As a consequence, when development proceeds the patches of'new' cell membrane cover most of the cell surface, leaving only the outer surfaceof the peripheral blastomeres wrapped in 'old' cell membrane.

This interpretation suggests that 'new' membrane is formed at the edge of thefurrow, either by the local assembly of components or by the fusion there ofpreassembled components (see Doyle & Bauman, 1979). Vesicles associating

FIGURES 22-25

Fig. 22. Advanced morula (80 h of development). The cell surface of the embryobeneath the zona pellucida (zp) shows no APA, while the activity is strong betweenblastomeres.Fig. 23. Early blastocyst (84 h of development). No APA is detected on the cellmembranes facing the perivitelline space (pvs) while a strong activity is observedbetween blastomeres. Some crystalloid bodies in the cytoplasm (cb) show no enzymeactivity.Fig. 24. Early blastocyst (84 h of development). The cell membranes bordering theperivitelline space (pvs) show no APA. The activity appears beneath the apicaljunction, (arrow).Fig. 25. Early blastocyst (84 h of development). No APA is detected on the cellmembrane bordering the perivitelline space (pvs) nor the blastocoelic cavity (be),while activity between blastomeres is strong.


with the cleavage furrow have been described in embryos of a species of poly-chaete (Emanuelsson, 1974) and in the embryo of Xenopus (Sanders & Singal,1975) but in our material we have not observed APA in the cytoplasm near theplace where cell membrane is supposedly formed. Therefore, in order to upholdthis interpretation we would be led to assume that alkaline phosphatase isinactive until it reaches the outer surface of the cell membrane or that itsactivity requiies the kind of steric organization present at the cell membraneand in crystalloid bodies.

The interpretation based on the formation of 'new' cell membrane seems tobe inconsistent with the absence of APA on the blastocoel surafce. Mulnard &Huygens (1978) suggest that the enzyme there might be lost as a result of'desquamation' of the cell membrane, which would explain why they detectAPA on cellular debris in the blastocoel. Although we have also observed leadphosphate grains in the blastocoel, we have been unable to identify membranousdebris and therefore we would regard as a possibility that the lining of the blasto-coel derives from remnants of' old' cell membranes present between blastomeres.

Up to now we have disregarded any relation between APA patches andcrystalloid bodies, though these are the only two localizations of APA, becausethey are spatially apart; however, crystalloid bodies may well be stores ofcortical material, indirectly related to the cell membrane through the system ofmicrotubules and filaments. This beomes a distinct possibility when one con-siders: first, that the treatment of mouse cleaving embryos with Cytochalasin Bpromotes the deposition of crystalloid bodies (Moskalewski, Sawicki, Gabara& Koprowski, 1972; Perry & Snow, 1975); second, that crystalloid bodies intheir highly structured form look like packages of filaments or microtubules(Daniel & Kennedy, 1978); and third, that cleaving mouse embryos synthesizeactin and tubulin at a very high rate (Abreu & Brinster, 1978).

Now, microtubules and filaments may be involved in several ways in theregionalization of the cell membrane. If APA patches turn out to be lots of'new' membrane, microtubules and filaments would be required to preventalkaline phosphatase from diffusing in the plane of the fluid membrane;moreover, the enzyme molecules on the surface might correspond to the ends ofmicrotubules or filaments inserted in the membrane.

However, a good morphological correlation of APA patches with cleavagefurrows and the interblastomeric surfaces does not exclude the possibility thatpatches are formed by entirely different mechanisms, such as those responsiblefor lymphocyte capping (Edelman, 1976). Actually, since the forces shaping theembryo are exerted by the cortical complex of microtubules and filaments, theirlocal concentration at the cleavage furrows and at the inner side of blastomeresduring compaction might account for the distribution of APA patches. Adisturbance of such cortical pattein, when morulae are dissociated and cultured,may explain the somewhat inconstant and variable distribution of APA patchesin the artificially produced outer surfaces.


The morphogenetic effect of a cell membrane regionalization during mam-malian cleavage has been discussed elsewhere (Izquierdo, 1977); we shouldonly emphasize here that a decisive morphogenetic step leading from morulaeto blastocysts is the establishment of zonular tight junctions (Ducibella,Albertini, Anderson & Biggers, 1975; Izquierdo, Fernandez & Lopez, 1976),and our results suggest that these are formed around the peripheral cells of theembryo, precisely where the cell membrane region, which exhibits APA, meetsthe region which does not.

This research was supported by grant RLA 78/024 from a PNUD/UNESCO programmeand by grant no B-256-781 from the University of Chile.

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(Received 17 October 1979, revised 17 April 1980)

cell membrane regions in preimplantation mouse …cell membrane regions in preimplantation mouse...

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