/ . Embryol. exp. Morph. Vol. 59, pp. 89-102, 1980
Printed in Great Britain © Company of Biologists Limited 1980
89
Cell membrane regions in preimplantation
mouse embryos
By L. IZQUIERDO 1 , T. LOPEZ 1 AND P. MARTICORENA 1
From the Department of Biology, Faculty of Science,
University of Chile
SUMMARY
Cell membrane regions characterized by alkaline phosphatase activity are described in
cleaving mouse embryos and early blastocysts. Enzyme activity is demonstrated by light and
electron microscopy, from the late 4-cell stage onwards, on the cell surfaces between blastomeres but not on the outer surface of the embryo. Experiments with dissociated morulae
show that this is probably not an artifact due to the retention of the enzyme reaction product
between the blastomeres. With the electron microscope the activity is also demonstrated in
crystalloid bodies within the cytoplasm. The localization and growth during cleavage of cell
membrane regions with enzyme activity is interpreted as the result of new cell membrane
formation and/or as a relation of the crystalloid bodies with the cell membrane through the
cortical system of microtubules and filaments.
INTRODUCTION
Since the first demonstration by Mulnard (1955) of non-specific alkaline
phosphatase activity (APA) in early mammalian embryos, several research
groups have been attracted to the subject because the onset of the enzyme
activity and its localization suggest a pattern of cell differentiation and morphogenesis (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 direct
biochemical assay showed the sudden appearance of the enzyme activity in
8-cell mouse embryos. For further precision, individual embryos were tested
by means of a cytochemical method and the enzyme activity was demonstrated
in some late 4-cell embryos (Izquierdo & Marticorena, 1975). Observations
on mouse, rat and hamster embryos, as well as on mouse embryos cultured
in vitro, confirmed and extended our previous cytochemical results (Ishiyama
& Izquierdo, 1977).
As far as the localization of APA in morulae and blastocysts is concerned
1
Authors' address: Department of Biology, Faculty of Science, University of Chile,
Casilla 653, Santiago, Chile.
90
L. IZQUIERDO, T. LOPEZ AND P. MARTICORENA
20/i/m
20 nm
>*~s
30jum
80 nm
15 ^m
10/im
Cell membrane regions in preimplantation mouse embryos
91
reports are at variance on whether inner and outer cells stain differentially.
These contradictory observations have been ascribed to artifacts caused either
by unreliable cytochemical methods or by the unequal thickenss of the embryos,
when examined in whole mounts (Wilson, Bolton & Cuttler, 1972; Solter et ah
1973). However, recent reports on APA localization are largely in agreement.
Electron microscope observations of mouse morulae and blastocyst stained by
various modifications of the Gomori-Takamatsu method show that APA is
confined to the cell surface between blastomeres and to crystalloid bodies
found in the cytoplasm, and is detected neither at the outer surface of the
embryo nor at the lining of the blastocoel (Izquierdo, 1977; Mulnard & Huygens, 1978). Light microscope observations on mouse embryos stained by an
azo-dye cytochemical method show a somewhat similar distribution of APA
(Johnson et al. 1977). The disparate results of Vorbrodt et al. (1977) have
been attributed to unspecific staining produced by long incubation at a relatively
low pH (Mulnard & Huygens, 1978).
In this paper we deal with the possibility of an artifact due to the retention of
the enzyme reaction product between the blastomeres, then we describe the
regionalization of the cell membrane as revealed by APA and finally we discuss
the 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 is
recognized.
Fig. 2. Whole mount of an 8-cell stage (57 h of development). APA is observed
between blastomeres.
Fig. 3. Whole mount of two morulae of about 10 cells (60 h of development). Embryo
at the left side reveals APA, embryo at the right side is a control incubated without
substrate.
Fig. 4. Whole mount of a pair of blastomeres isolated from a morula of about 10
cells (60 h of development) and immediately processed for APA demonstration. APA
patches are observed between blastomeres and on the surfaces exposed by dissociation (arrows).
Figs. 5 and 6. A whole mount of one pair of blastomeres isolated from a morula of
about 12 blastomeres (61 h of development) photographed in two positions. After
their isolation from the morula the pair was cultured in vitro for 17 h before fixation
and APA demonstration. An APA patch is shown between the blastomeres, but no
other patches are observed on the surface.
Fig. 7. Whole mount of a morula of about 16 cells (64 h of development), halved in
two parts and cultured in vitro for 15 h before fixation and APA demonstration.
The part represented shows APA between the compacted blastomeres and a patch
on the surface exposed by halving (arrows).
Fig. 8. Whole mount of a 2-cell stage (38 h of development) fused to a morula of
about 16 cells (62 h of development) and cultured in vitro for 5 h before fixation and
APA demonstration. No APA is observed in the 2-cell stage nor in its contact with
the morula. The morula shows the typical localization of APA at this stage.
92
L. IZQUIERDO, T. L6PEZ, AND P. MARTICORENA
T'
.11
Cell membrane regions in preimplantation mouse embryos
93
cleavage an intrinsic spatial pattern gradually unfolds, providing cells with
information about their position in the embryo (Izquierdo, 1977).
MATERIALS AND METHODS
Preimplantation embryos were obtained from natural matings of SwissRockefeller mice. Their stage is indicated in number of cells or in hours of
development, reckoned since the beginning of the day when the vaginal plug is
found.
The embryos recovered from the oviduct were either immediately processed
for APA demonstration or they were first treated so as to dissociate blastomeres
or associate embryos. To this end, eggs were denuded of the zona pellucida
by pipetting them in 5 % Pronase (Calbiochem) in Biggers's culture medium
(Biggers, Whitten & Whittingham, 1971) devoid of albumin, for 4 min at room
temperature; the embryos were then rinsed in Biggers's medium and manipulated under mineral oil with fluid jets produced by a pipette and with the help
of gentle touches. Thereafter they were either fixed, or cultured in vitro before
fixation. Fixation lasted for 60 min in cold 3 % glutaraldehyde in 0-05 M
cacodylate buffer at pH 7-4. After fixation, embryos were rinsed in buffer for
30 min and incubated for 90 min at room temperature in an alkaline lead
citrate medium at pH 9-5 (Mayahara, Hirano, Saito & Ogawa, 1967). Best
results were obtained when the medium was buffered with tris-maleic acid and
stabilized with 8 % glucose (Sundstrom & Mornstadt, 1975).
For light microscopy, after incubation the embryos were rinsed in buffer and
the enzyme reaction product was developed with ammonium sulphide. The
embryos were then immersed in glycerol and whole mounts observed under
oblique illumination so as to cast shadows (Hlinka & Sanders, 1970) which
clearly reveal the contours of the nuclei and blastomeres, though this illumination somewhat hinders an accurate recognition of APA localization on black
and white photographs.
For electron microscopy, embryos were rinsed in buffer (pH 7-4) and postfixed for 60 min in 1 % OsO4 in cacodylate buffer; then they were dehydrated in
acetone and embedded in low-viscosity resin (Spurr, Polyscience). Thin sections
F I G U R E S 9—14
Figs. 9-11. Morulae of 8 cells (60 h of development). The reaction product accumulates on the outer surface of the plasma membrane at the edge of the cleavage
furrows.
Figs. 12 and 13. Morula of 8 cells (60 h of development). Discrete patches of APA
on the surface of adjoining cells. Fig. 12 shows that the perivitelline space (pvs) is
continuous with the interblastomeric cleft.
Fig. 14. Morula of about 16 cells (65 h of development). Extended APA patches on
the cell membrane between blastomeres. APA is also detected in crystalloid bodies
(cb) within the cytoplasm.
7
EMB
59
94
L. IZQUIERDO, T. LOPEZ AND P. MARTICORENA
I*
•
Cell membrane regions in preimplantation mouse embryos
95
were stained with uranyl acetate and observed in a Philips EM 300 electron
microscope.
Controls were performed on embryos whose enzyme was heat-denaturated
after 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 of
approximately 300 embryos studied, 45 were observed with the electron microscope.
RESULTS
Light microscopy of whole embryos and electron microscopy of thin sections
confirm previous observations in that no APA is detected prior to the late 4-cell
stage (see Introduction). The results concerning the localization of APA in
later stages of development will be summarized in this order: light microscope
observations on intact embryos and on manipulated embryos, and observations
with the electron microscope.
Light microscopy. From the late 4-cell stage onwards (Fig. 1) while APA is
neatly observed on the cell surface between blastomeres, no activity is detected
on the outer surface of the embryos nor is it possible to affirm that there is
activity on cytoplasmic structure (Figs. 2, 3). If embryos are heated after
fixation 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 APA
demonstration and observed in whole mounts, interblastomeric patches are
evident and APA patches can also be recognized on the cell surface that has
become outer surface by virtue of the dissociation procedure (Fig. 4). If the
dissociated pairs are cultured overnight before APA demonstration, the outer
patches are not always evident (Figs. 5, 6); actually, they are more clearly seen
if morulae were dissociated into portions with several blastomeres each (Fig. 7).
The variability of these results and the limited number of observations do not
allow a systematic description, but they support the reality of outer APA patches
on the cell surface exposed by dissociation.
FIGURES
15-19
Fig. 15. Morula of about 16 cells (65 h of development). Discrete APA patches observed on the cell surface between three blastomeres.
Fig. 16. Morula of about 16 cells (65 h of development). Typical crystalloid bodies
in the cytoplasm showing APA.
Fig. 17. Advanced morula (80 h of development). A wide interblastomeric space
shows APA on both cell surfaces.
Fig. 18. Advanced morula (80 h of development). The nucleolus (nu) and cytoplasmic 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.
7-2
96
L. I Z Q U I E R D O , T. LOPEZ AND P. MARTICORENA
20
Figs. 20 and 21. Advanced morulae (80 h of development). APA is detected on
the surface of blastomeres and in crystalloid bodies (cb). No APA is detected
in the nucleolus (nu) nor in mitochondria nor other cytoplasmic organelles, except
crystalloid bodies.
Cell membrane regions in preimplantation mouse embryos
97
By fusing synchronous or asynchronous morulae in pairs we tried to convert
a part of their outer surface, which never reveals APA, into interblastomeric
surface, which always does. After 5 h in culture the fused embryos were processed
for APA demonstration and observed in whole mounts. Under these conditions
the artificial interblastomeric surfaces do not reveal any APA (Fig. 8).
Electron microscopy. In early morulae, APA is localized on the cell membrane
both at the advancing edge of the cleavage furrows (Figs. 9-11) and in discrete
patches on adjoining blastomers (Figs. 12, 13). In late morulae and blastocysts,
APA patches have spread out, and comprise most of the cell membrane bordering the interblastomeric clefts (Figs. 18, 20-25).
Although the cytochemical procedure interferes with a clear definition of the
cell membrane, the enzyme reaction product appears to be lumped on the outer
surface while the inner surface shows a smooth profile. Narrow interblastomeric
spaces usually appear full of lead phosphate, but when the spaces are sufficiently
wide, the reaction product may be detected on both cell membranes or on
neither of them, as if cell membranes facing each other were symmetrical
regarding their APA localization (Figs. 17-19).
In all developmental stages studied, the cell membranes around the embryo
appear to be devoid of APA. The transition from the outer inactive region to the
active region between blastomeres is quite abrupt, even in early morulae when
apical tight junctions are still absent (Fig. 12). The cell surface bordering the
blastocoelic cavity lacks APA, and as a consequence APA patches are exhibited
exclusively between trophectoderm cells (Fig. 23). Lead phosphate grains are
usually found scattered in the blastocoel associated with cellular debris which
do not reveal clear-cut membrane structures.
The membrane of the many diverse cytoplasmic organelles does not show
APA, and this is also the case for non-membranous structures except for
crystalloid bodies, which are known to arise during cleavage in mammalian
embryos of several species (see Calarco & Brown, 1969; Van Blerkom, Manes &
Daniel, 1973). In the mouse embryo, crystalloid bodies appear as structures of
approximately 0-5 /«n in diameter that reveal a substructure periodicity of
120-130 nm in the tightly packed crystalline form. These structures may also be
recognized in diverse transitional forms, from typical crystalloid bodies to a
network of loosely associated cross-striated microtubules or filaments.
Crystalloid bodies may show a strong APA (Figs. 14, 16, 21) or no activity
at all (Fig. 23), even at the same stage of development; the less structured forms
do not show APA. We have not observed an association of crystalloid bodies
with the APA patches nor with any other part of the cell membrane.
98
L. IZQUIERDO, T. LOPEZ AND P. MARTICORENA
pvs
24
Cell membrane regions in preimplantation mouse embryos
99
DISCUSSION
Mulnard & Huygens (1978) analysed several possible causes of false localization of APA in preimplantation embryos but they did not consider an eventual
artifact caused by the retention of the enzyme reaction product in the narrow
clefts between blastomeres while it freely diffuses out from the surface around
the embryo and from the surface bordering the blastocoel. In fact, lead phosphate is insoluble in our experimental conditions; however, this artifact would
closely mimic the APA distribution we describe in intact embryos. The presence
of APA patches on the new outer surface produced by dissociating morulae and
the absence of APA patches on the new interblastomeric surface produced by
fusing embryos is inconsistent with an artifact due to differential retention of
lead phosphate, though further investigation may be required to dismiss it
entirely. On these bases we submit that the pattern of APA distribution in
preimplantation embryos reveals a true regionalization of the cell membrane
that deserves to be discussed.
Considering first, that a large supply of cell membrane is needed during cleavage; second, that APA is detected on the cell membrane (Izquierdo, 1977;
Mulnard & Huygens, 1978; this paper); third, that the activity only arises at
the late 4-cell stage (Izquierdo & Marticorena, 1975; Ishiyama1 &} Izquierdo,
1977) one would expect to recognize 'new' cell membrane if it] is] added in
discrete lots to the cell surface (Izquierdo, 1977). Indeed, our observations
suggest that 'new' membrane is formed at the leading edge of the cleavage
furrow, leaving symmetrical patches between blastomeres when their division
is completed. As a consequence, when development proceeds the patches of
'new' cell membrane cover most of the cell surface, leaving only the outer surface
of the peripheral blastomeres wrapped in 'old' cell membrane.
This interpretation suggests that 'new' membrane is formed at the edge of the
furrow, either by the local assembly of components or by the fusion there of
preassembled components (see Doyle & Bauman, 1979). Vesicles associating
FIGURES
22-25
Fig. 22. Advanced morula (80 h of development). The cell surface of the embryo
beneath the zona pellucida (zp) shows no APA, while the activity is strong between
blastomeres.
Fig. 23. Early blastocyst (84 h of development). No APA is detected on the cell
membranes facing the perivitelline space (pvs) while a strong activity is observed
between blastomeres. Some crystalloid bodies in the cytoplasm (cb) show no enzyme
activity.
Fig. 24. Early blastocyst (84 h of development). The cell membranes bordering the
perivitelline space (pvs) show no APA. The activity appears beneath the apical
junction, (arrow).
Fig. 25. Early blastocyst (84 h of development). No APA is detected on the cell
membrane bordering the perivitelline space (pvs) nor the blastocoelic cavity (be),
while activity between blastomeres is strong.
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L. IZQUIERDO, T. LOPEZ AND P. MARTICORENA
with the cleavage furrow have been described in embryos of a species of polychaete (Emanuelsson, 1974) and in the embryo of Xenopus (Sanders & Singal,
1975) but in our material we have not observed APA in the cytoplasm near the
place where cell membrane is supposedly formed. Therefore, in order to uphold
this interpretation we would be led to assume that alkaline phosphatase is
inactive until it reaches the outer surface of the cell membrane or that its
activity requiies the kind of steric organization present at the cell membrane
and in crystalloid bodies.
The interpretation based on the formation of 'new' cell membrane seems to
be 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 detect
APA on cellular debris in the blastocoel. Although we have also observed lead
phosphate grains in the blastocoel, we have been unable to identify membranous
debris and therefore we would regard as a possibility that the lining of the blastocoel derives from remnants of' old' cell membranes present between blastomeres.
Up to now we have disregarded any relation between APA patches and
crystalloid bodies, though these are the only two localizations of APA, because
they are spatially apart; however, crystalloid bodies may well be stores of
cortical material, indirectly related to the cell membrane through the system of
microtubules and filaments. This beomes a distinct possibility when one considers: first, that the treatment of mouse cleaving embryos with Cytochalasin B
promotes the deposition of crystalloid bodies (Moskalewski, Sawicki, Gabara
& Koprowski, 1972; Perry & Snow, 1975); second, that crystalloid bodies in
their highly structured form look like packages of filaments or microtubules
(Daniel & Kennedy, 1978); and third, that cleaving mouse embryos synthesize
actin and tubulin at a very high rate (Abreu & Brinster, 1978).
Now, microtubules and filaments may be involved in several ways in the
regionalization of the cell membrane. If APA patches turn out to be lots of
'new' membrane, microtubules and filaments would be required to prevent
alkaline phosphatase from diffusing in the plane of the fluid membrane;
moreover, the enzyme molecules on the surface might correspond to the ends of
microtubules or filaments inserted in the membrane.
However, a good morphological correlation of APA patches with cleavage
furrows and the interblastomeric surfaces does not exclude the possibility that
patches are formed by entirely different mechanisms, such as those responsible
for lymphocyte capping (Edelman, 1976). Actually, since the forces shaping the
embryo are exerted by the cortical complex of microtubules and filaments, their
local concentration at the cleavage furrows and at the inner side of blastomeres
during compaction might account for the distribution of APA patches. A
disturbance of such cortical pattein, when morulae are dissociated and cultured,
may explain the somewhat inconstant and variable distribution of APA patches
in the artificially produced outer surfaces.
Cell membrane regions in preimplantation mouse embryos
101
The morphogenetic effect of a cell membrane regionalization during mammalian cleavage has been discussed elsewhere (Izquierdo, 1977); we should
only emphasize here that a decisive morphogenetic step leading from morulae
to 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 the
embryo, precisely where the cell membrane region, which exhibits APA, meets
the region which does not.
This research was supported by grant RLA 78/024 from a PNUD/UNESCO programme
and by grant no B-256-781 from the University of Chile.
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(Received 17 October 1979, revised 17 April 1980)