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Surface coats of the mouse blastocyst and uterus during the preimplantation period.

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Surface Coats of the Mouse Blastocyst and Uterus
during the Preimplantation Period ’
ALLEN C. ENDERS AND SANDRA SCHLAFKE
Department of Anatomy, Washington University School of Medicine,
St. Louis, Missouri 631 10
ABSTRACT
A glycoprotein coat is demonstrable on the free surface of both
the blastocyst and uterine luminal epithelium of the mouse on day 4 and day 5
of normal pregnancy, and on day 7 of delayed implantation, using concanavalin
A-peroxidase and ruthenium red. The coats are apparently negatively charged,
as shown’by their binding with colloidal thorium dioxide. The cell coat on uterine
epithelium is appreciably thicker than that on the blastocyst. The information
currently available is sufficient to suggest that simplistic mechanisms such as
change in charge or total thickness cannot be the sole basis of initial adhesion,
but that some localized reduction of the uterine surface coat accompanies ad-
hesion. However, considerably more information is necessary concerning the
nature of the surface coats before a more comprehensive understanding of the
role of adhesion in implantation can be achieved.
The process of implantation of the blastocyst in the endometrium that results in
the establishment of a hemochorial placenta necessarily involves cellular interactions between the trophoblast of the
blastocyst and the luminal epithelium of
the uterus. Since the first aspect of the
cells to contact would be expected to be
the surface coats, it is not surprising that
the surface coats have finally begun to receive attention in recent studies of implantation. The mouse blastocyst, like that
of the rat, is relatively passive in the first
stages of implantation (Enders, ’72). The
blastocyst can be flushed readily from the
developing implantation site when first
“clasped by the uterus, However, within
a day, the blastocyst adheres to the uterine
epithelium along limited portions of the
trophoblast cell surface sufficiently firmly
to resist displacement of the blastocyst during flushing of the uterus with fixative. In
addition to direct adhesion to the luminal
epithelium, blastocysts are capable of
phagocytizing sloughed epithelial cells
(Finn and Lawn, ’68) well before penetration of the residual basal lamina of the
uterine epithelium. Consequently these
events involving adhesion, together with
giant cell formation (Dickson, ’66), are
chronologically separated from active invasion of the decidua.
ANAT. REC.,180: 31-46.
Previous studies of the surface coats of
the mouse blastocyst have used either
whole mounts or extraction techniques.
Holmes and Dickson (’73) used Prussian
Blue staining of colloidal iron in a n attempt to demonstrate surface coats by light
microscopy using whole mounts of mouse
blastocysts. Since the blastocyst did not
stain heavily with this method until after
the estrogen surge, they concluded that
there is a “change in the amount or functional activity of the surface glycoproteins”
of the blastocyst at this stage. Pinsker and
Mintz (’73) exposed mouse preimplantation stages in vitro to trypsin after previous
incubation with radioactive glucosamine.
Since the cleavage stages and blastocysts
were “still viable” after exposure to trypsin,
they concluded that anything removed by
the trypsin was from the surface. The
trypsinate thus removed (and subsequently
digested for five days with pronase) showed
a n increase in incorporation of glucosamine and its derivatives, and also a n increase in molecular weight of the polysaccharide units in morulae and early
blastocysts a s opposed to 2 to 4-cell stages.
The present study was initiated to demonstrate directly the surface coats of the
Received Jan. 8, ’74. Accepted Feb. 5, ’74.
1 Supported by grant 2 R01 HD04962 from the National Institute of Child Health and Human Development.
31
32
ALLEN C. ENDERS AND SANDRA SCHLAFKE
mouse blastocyst and uterus as a preliminary step in the analysis of these coats and
their relationship to the events of implantation.
MATERIALS AND METHODS
Collection of stages
Young adult female mice (Swiss albino)
were housed four to a cage. An adult male
mouse was placed in each cage in the early
evening, and the females were checked
the next morning for the presence of a
vaginal plug. The day on which a plug was
found was designated day 1. Embryos were
collected at two times on day 4 (9 :00 AM
and 4:OO P M ) , on day 5 (9:OO-11:OO AM)
and on day 7 of lactationally delayed implantation (induced by the post partum
mating of females suckling 7-10 young).
tive in 0.1 M cacodylate buffer to a final
concentration of 0.05% (Luft, '71). Tissues were fixed in this solution for 60 minutes at room temperature and rinsed in 0.1
M cacodylate buffer. The same concentration of ruthenium red was present in the
subsequent fixative, 2% osmium tetroxide
in 0.1 M s-collidine buffer, pH 7.3. Following postfixation for 60-120 minutes, the
tissues were rapidly dehydrated and
embedded.
Thorium dioxide
After both initial fixation in aldehyde
fixative in phosphate buffer and postfixation in osmium in phosphate buffer, tissues
were rinsed in 3% acetic acid, then incubated in 1% thorium dioxide (Thorotrast,
Testagar) in 3% acetic acid for 18 hours
at room temperature (Rambourg and
Concanavalin A method
Leblond, '67). They were then rinsed
Blastocysts and transverse slices of the
mid-region of the uterus were fixed for 60 briefly in 3% acetic acid, dehydrated and
minutes in 2% formaldehyde-2
1/2% embedded. In one instance blastocysts and
glutaraldehyde in 0.1 M phosphate buffer, uterus were methylated in 4% thionyl
pH 7.3. After a rinse in buffer, tissues were chloride in absolute methanol (Stoward,
incubated in concanavalin A (Sigma '67) for 6 hours at room temperature,
Grade IV) at a concentration of 1 mg/ml rinsed, postfixed, then treated with thorium
for 30 minutes at room temperature dioxide as above.
Two to five blastocysts and transverse
(Bernhard and Avrameas, '71). Following
an additional buffer rinse, the tissues slices of uteri were used from 7 mice on
were incubated in horseradish peroxidase day 4, 11 mice on day 5, and 6 mice on
(Sigma Type 11) at a concentration of 1 day 7 of delayed implantation.
mg/ml for 30 minutes at room temperaOBSERVATIONS
ture, thoroughly rinsed, and placed in diBlastocyst
aminobenzidine-H202medium for 15 minutes (Graham and Karnovsky, '66). All
tissues were then postfixed in 2% osmium Concanavalin A procedure
A distinct surface coat can be demontetroxide in 0.1 M phosphate buffer for 60
minutes at 4"C, dehydrated rapidly in cold strated by the concanavalin A-peroxidase
alcohol, and embedded in Durcupan epoxy method on the apical surface of trophoresin. Blastocysts and uteri from each time blast cells of all stages examined (figs.
period were incubated similarly but with 1-3, 6, 7). ConA stains the zona pellucida
0.2 M a-methyl-D-mannoside (Sigma) as well as extracellular material in the
added to the concanavalin A and peroxi- space between the zona and trophoblast on
dase solutions, serving as controls for non- day 4 , 9 :00 AM. Some of the blastocysts on
specific adsorption of concanavalin A. Ad- day 4, 4 : O O PM, have lost their zonas, but
ditional specimens incubated in peroxidase there does not appear to be any difference
after fixation but without exposure to con- in coat between those that have lost their
canavalin A served as a control for non- zonas and those that have not. In all of
the day 4 blastocysts, there is an unevenspecific adsorption of peroxidase per se.
ness of the coat, in that pockets with a
Ruthenium red procedure
thicker layer alternate with regions of reRuthenium red (Electron Microscopy duced thickness. The ConA penetrates beSciences) was added to the aldehyde fixa- tween trophoblast cells only to the apical
SURFACE COATS OF MOUSE BLASTOCYST
junctional complex, where it stops abruptly
(fig. 2).
The ConA coat on the day 5 blastocyst
is uniform and moderately thick, and as
in the previous stage, stops abruptly at the
junctional complexes. However, at this
stage the apical junctions are directly at
the surface or even in a protruding ridge,
in contrast to the previous stage where the
effective junction is below the general level
of the blastocyst surface (figs. 3, 6).
Frequently two or three trophoblast cells
of zona-free blastocysts are damaged by the
ConA and peroxidase procedure, permitting access of the stain complex to the interior, In these blastocysts, the basal
lamina beneath the trophoblast stains very
heavily, as does intercellular material up to
the level of the apical junctional complex
(figs, 6, 7). Interestingly the endodermal
cells of the embryonic cell mass have only
a very thin coat (fig. 6 ) . Other cells of the
embryonic cell mass also appear to have a
thin coat, but it is not always clear that
access to the ConA was as complete for
these cells as it is for the endodermal cells.
No consistent or appreciable difference in
apical cell coat thickness is seen with
these methods between the trophoblast
layer overlying the embryonic cell mass
(Rauber’s layer) and the abembryonic
trophoblast. In light micrographs of whole
ConA-treated blastocysts, the damaged cells
are clearly visible, and their apparently
random distribution in the trophoblast can
be seen. However, the surface coats cannot
be discerned without sectioning.
The distribution and thickness of the
surface coat is essentially the same on
blastocysts during delayed implantation
(day 7 ) as on the day 5 blastocyst, although the basal lamina of delay blastocysts is thicker and more heavily stained
(fig. 7).
In all instances the ConA staining is
eliminated by inclusion of a-methy1-Dmannoside (which binds to the active sites
on the ConA molecule) with the ConA and
peroxidase incubations (fig. 4 ), Although
blastocysts incubated in ConA and peroxidase in the presence of the mannoside appeared to darken in the diaminobenzidine
incubation, no surface coat could be demonstrated in electron micrographs of these
blastocysts.
33
Ruthenium red procedure
Only a thin coat is seen at the free surfaces of trophoblast cells of blastocysts at
the three stages examined with the ruthenium red method (fig. 5). Ruthenium red
stains material between trophoblast cells
and also the basal lamina, However, ruthenium red frequently causes extensive damage to several trophoblast cells in a blastocyst, diminishing the suitability of this
method for distinguishing regional differences in coat distribution.
Colloidal thorium
Colloidal thorium stains a thin layer at
the surface of all of the blastocysts examined. Slightly more thorium is seen on
the surface of the delay blastocysts than
on that of the earlier stages (figs. 8, 9).
Methylation prior to thorium staining
results in poor tissue preservation, but
clearly eliminates the binding of the positive colloid to the surface coat. However
in the case of zona-surrounded blastocysts,
it is not certain that the thorium penetrates
the zona after methylation.
Uterine luminal epithelium
Surface coats on the apical surface of
luminal epithelial cells from the lateral
borders of the uterine lumen are readily
demonstrated with all three methods (figs.
10-15). However, both ruthenium red and
especially ConA stain irregular patches of
luminal cells. Since the ConA-peroxidase
reaction product is consistently absent
from necks of glands, folds, and even shallow pockets, it is concluded that penetration at one of the stages of preparation is
the principal problem, preventing more
uniform distribution within the uterus.
The surface coat is thinnest and most
uniform with the colloidal thorium method
(fig. 15). Both the ConA and ruthenium
red methods demonstrate a coat on the
uterine luminal epithelial cells that is substantially thicker than that on the blastocyst (figs, 10-12, 1 4 ) . Little difference in
the nature of the coat between the stages
examined could be demonstrated using
these three methods. However, it should be
noted that no attempt was made to examine the actual site of location of the
blastocyst in the uterus. The methods as
applied demonstrate the essential similar-
34
ALLEN C. ENDERS AND SANDRA SCHLAFKE
cellular adhesion during development have
been rapid. Roseman ('70) and Roth et al.
('71) introduced an interesting theory in
which it was suggested that the glycosyltransferases form the bridge molecules between cells by attaching to their appropriDISCUSSION
ate substrate sugars in the adjacent cell
Although the surface coat of the mouse coat. Recently Chipowsky et al. ('73) have
blastocyst is not especially thick, i t can be shown that cultured fibroblasts will adhere
expected to be very complex. Consequently to insoluble analogs of selected cell surface
the methods used here constitute only a carbohydrates. Weisman et al. ('72) demsmall beginning in the characterization of onstrated that more cohesive cells exclude
this material.
less cohesive cells in tissue assembly patThe staining by ruthenium red indicates terns. Weiser ('73) has shown that the
the presence of acidic polysaccharides at surfaces of less mature cells of intestinal
the cell surface (Luft, '71). The binding crypts will incorporate sugar nucleotides
of concanavalin A and the abolition of this that the more mature cells on the villi will
binding by a-methyl-D-mannoside astab- not.
Despite these selected examples and
lishes the presence of saccharides in the
coat. Although concanavalin A preferen- many other excellent studies of surface
tially binds to mannose, it can also bind interactions, no single complete molecular
with lesser affinity to a series of other mechanism of adhesion between normal
sugars, so it is not by itself specific for this cells has been worked out. Since we know
group (Iyer and Goldstein, '73). Further- even less about the cell coats in the admore, since it is the reaction product from hesion of trophoblast to uterine cells than
incubation (for a standard 15 min) that we do in the cases cited above, it would be
is visualized, thickness bears only a crude premature to expound an extensive theory
relation to initial ConA binding. The bind- of coat interaction at implantation. Nevering of colloidal thorium dioxide and the theless a few aspects of this association
abolition of this binding by prior methyla- can be clarified.
It is extremely unlikely, for example,
tion is an indication of the presence of
acidic carbohydrates at the surface (Ram- that the adhesion of blastocyst to uterus is
bourg and Leblond, '67; Stoward, '67).
a simple matter of coat charge. As previSince the colloidal thorium method dem- ously mentioned, both blastocyst and uteronstrated acidic groups on the surface of ine coats appear similar in their reactivity
all blastocyst stages examined, it seems both to ruthenium red and to colloidal
probable that the colloidal iron staining re- thorium. Consequently, electrostatic forces
ported by Holmes and Dickson ('73) for between the surfaces would tend to be reblastocysts only after the estrogenic surge pulsive. This is not surprising, since most
reflects other conditions in the whole mammalian cells do carry a net negative
mounts rather than being due solely to charge (Weiss and Zeigel, '71). It is preanionic groups at the surface. Other stud- sumably such charges that are overcome
ies suggesting the presence of anionic both by natural adhesion and by experigroups on the surface of mammalian eggs mental adhesion such as that produced by
have been those of Yanagimachi et al. some of the phytohemagglutinins (Noonan
('73) using colloidal iron hydroxide to in- and Burger, '73). Clemetson et al. ('72,
dicate a negative charge on the hamster '73) have suggested that changes in the K+
egg at fertilization and of Cooper and Bed- content of uterine fluid might lead to a
ford ('71) using a similar method on rab- decrease in negative membrane potential
bit eggs at fertilization. In addition, of the trophoblast, thus facilitating adClemetson et al. ('71) demonstrated a neg- hesion. Jenkinson and Wilson ('73) have
ative surface charge on the rat blastocyst pointed out however that trophoblast can
after loss of the zona pellucida using elec- become adhesive in vitro in the absence of
trophoretic methods.
changes in the Na/K ratio in the medium.
In recent years, progress in studies of
Studies with adhering and non-adhering
ity of the surface coats of the uterus to
those of the blastocyst, with the exception
that the apical coat of uterine luminal epithelial cells is substantially thicker than
the coat of trophoblast cells.
SURFACE COATS OF MOUSE BLASTOCYST
cells have yielded mixed results with regard to cell coat thickness. Mitotic cells
(weakly adhering) generally have thicker
cell coats (Fox et al., '71 ). So also do many
transformed cells which similarly are nonadhering (Poste, '73). On the other hand,
in some instances conversion of non-adhesive to adhesive cells requires synthesis
involving saccharides (Openheimer et al.,
'69). A further indication that thckness
per se may not be significant is the observation of Glick et al. ('73) that differences
in the glycoprotein composition between
normal and transformed cells could be
found only in the deeper portions of the
coat.
In the rat and mouse, close apposition of
cell membranes occurs during adhesion of
blastocyst to uterus (Enders and Schlafke,
'67, '69; Potts, '68). This apposition indicates either a reduction in coat thickness
or an interaction such that the thickness
of the two coats involved is not summed.
Furthermore, Enders and Schlafke ('72)
illustrated that in the ferret there is a
specific removal of the uterine coat by
specialized regions of adjacent trophoblast.
At the present incomplete state of our
information, i t seems most likely that the
progressive localized adhesion in the
mouse and rat will be found to involve,
among other things, digestion of the coat
on the maternal surface to permit both
closer approximation and possibly the exposure of app.ropriate reactive groups on
the uterine surface. Further studies concerning the nature of the glycoprotein coat
of both trophoblast and uterine epithelial
cells, and determination of the presence
or absence of specific glycosyltransferases
at the surfaces of these cells should make
i t possible to begin to understand the adhesion stage of implantation.
The finding that neither ruthenium red
nor concanavalin A penetrate into the
apical junctional complexes indicates that
the two cell membranes are sufficiently approximated to be restrictive to molecules
with a diameter of 40 A or more. Other
types of studies would be necessary to determine whether a truly tight junction is
present in this location. It is interesting
that in blastocysts the cytoplasm surrounding the junction becomes raised on day 5,
and that the position of the junction moves
35
closer to the surface. Raised junctional
ridges have been reported previously by
scanning microscopy in the maturing
mouse blastocyst (Calarco and Epstein,
'73).
LITERATURE CITED
Bernhard, W., and S. Avrameas 1971 Ultrastructural visualization of cellular carbohydrate
components by means of concanavalin A. Exp.
Cell Res., 64: 232-236.
Calarco, P. G., and C. J. Epstein 1973 Cell
surface changes during preimplantation development in the mouse. Devel. Biol., 32: 208-213.
C,hipowsky, S . , Y. C. Lee and S. Roseman 1973
Adhesion of cultured fibroblasts to insoluble
analogues of cell-surface carbohydrates. Proc.
Nat. Acad. Sci., 70: 2309-2312.
Clemetson, C. A. B., M. M. Moshfeghi and V. R.
Mallikarjuneswara
1971 Surface charge o n
the five-day rat blastocyst. In: Biology of the
Blastocyst. R. J. Blandau, ed. University of Chicago Press, Chicago, pp. 193-205.
Clemetson, C. A. B., J. K. Kim, T. P. S. deJesus,
V. R. Mallikarjuneswara and J. H. Wilds 1973
Human uterine fluid potassium and the menstrual cycle. J. Obstet. Gynaecol. Brit. Comm.,
80: 553-561.
Clemetson, C. A. B., J. K. Kim, V. R. Mallikarjuneswara and J. H. Wilds 1972 The sodium
and potassium concentrations in the uterine
fluid of the rat at the time of implantation.
J. Endocr., 54: 417-423.
Cooper, C. W.,and J. M. Bedford 1971 Charge
density change in the vitelline surface following fertilization of the rabbit egg. J. Reprod.
Fert., 25: 431-436.
Dickson, A. D. 1966 The form of the mouse
blastocyst. J. Anat., 100: 335-348.
Enders, A. C. 1972 Mechanisms of implantation of the blastocyst. In: Biology of Reproduction. J. T. Velardo and B. Kasprow, eds., pp.
313-333.
Enders, A. C., and S. Schlafke 1967 A morphological analysis of the early implantation
stages in the rat. Am. J. Anat., 120: 185-226.
1969 Cytological aspects of trophoblastuterine interaction in early implantation. Am.
J. Anat., 125: 1-30.
1972 Implantation in the ferret: epithelial penetration. Am. J. Anat., 133: 291-313.
Finn, C. A,,and A. M. Lawn 1968 Transfer of
cellular material between the uterine epithelium and trophoblast during the early stages
of implantation. J. Reprod. Fert., 15: 333-336.
Fox, T. O., J. R. Sheppard and M. M. Burger
1971 Cyclic membrane changes in animal
cells: transformed cells permanently display a
surface architecture detected in normal cells
only during mitosis. Proc. Nat. Acad. Sci., 68:
244-247.
Glick, M. C., Y. Kimhi and U. Z. Littauer 1973
Glycopeptides from surface membranes of neuroblastoma cells. Proc. Nat. Acad. Sci., 70:
1682-1687.
Graham, R. C., and M. J. Karnovsky 1966 The
36
ALLEN C. ENDERS AND SANDRA SCHLAFKE
early stages of adsorption of injected horseradish peroxidase in the proximal tubules of
the mouse kidney: Ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem., 14: 291-302.
Holmes, P. V., and A. D. Dickson 1973 Estrogen-induced surface coat and enzyme changes
in the implanting mouse blastocyst. J. Embryol.,
exp. Morph., 29: 639-645.
Iyer, R. N.,and 1. J. Goldstein 1973 Quantitative studies on the interaction of concanavalin
A, the carbohydrate binding protein of the jack
bean, with model carbohydrate-protein conjugates. Immunochem., 10: 313-322.
Jenkinson, E. J., and I. B. Wilson 1973 In
vitro studies on the control of trophoblast outgrowth in the mouse. J. Embryol. exp. Morph.,
30: 21-30.
Luft, J. H. 1971 Ruthenium red and violet.
I. Chemistry, purification methods of use for
electron microscopy and mechanism of action.
Anat. Rec., 171: 347-368.
Noonan, K. D.,and M. M. Burger 1973 The
relationship of concanavalin A binding to
lectin-initiated cell agglutination. J. Cell Biol.,
59: 134-142.
Openheimer, S . B., M. Ediden, C. Orr and S . Roseman 1969 An L-glutamine requirement for
intercellular adhesion. Proc. Nat. Acad. Sci.,
63: 1395-1402.
Pinsker, M. C., and B. Mintz 1973 Change in
cell-surface glycoproteins of mouse embryos before implantation. Proc. Nat. Acad. Sci., 70:
1645-1648.
Poste, G. 1973 The synthesis of surface coat
materials in normal and transformed cells. Exp.
Cell Res., 77: 264-270.
Potts, D. M. 1968 The ultrastructure of implantation in the mouse. J. Anat., 103: 77-90.
Rambourg, A., and C. P. Leblond 1967 Electron
microscopic observations on the carbohydraterich cell coat present a t the surface of cells in
the rat. J. Cell Biol., 32: 27-53.
Roseman, S. 1970 The synthesis of complex
carbohydrates by multiglycosyltransferase systems and their potential function in intercellular adhesion. Chem. Phys. Lipids, 5: 270-297.
Roth, S., E. J. McGurie and S. Roseman 1971
Evidence for cell-surface glycosyltransferases.
Their potential role in cellular recognition.
J. Cell Biol., 51: 536-547.
Stoward, P. J. 1967 The histochemical properties of some periodate-reactive mucosubstances
of the pregnant Syrian hamster before and after
methylation with methanolic thionyl chloride.
J. Roy. Micr. SOC.,87: 77-103.
Weiser, M. M. 1973 Intestinal epithelial cell
surface membrane glycoprotein synthesis. 11.
Glycosyltransferases and endogenous acceptors
of the undifferentiated cell surface membrane.
J. Biol. Chem., 248: 2542-2548.
Weisman, O., M. Steinberg and H. Phillips 1972
Experimental modulation of intercellular cohesiveness: reversal of tissue assembly patterns.
Devel. Biol., 28: 498-517.
Weiss, L., and R. Zeigel 1971 Cell surface negativity and the binding of positively charged
particles. J. Cell Physiol., 77: 179-186.
Yanagimachi, R., G. L. Nicholson, Y. D. Noda
and M. Fujimoto 1973 Electron microscopic
observations of the distribution of acidic
anionic residues on hamster spermatozoa and
eggs before and during fertilization. J. Ultra.
Res.. 43: 344-353.
PLATES
PLATE 1
EXPLANATION OF FIGURES
1
Peroxidase reaction product is seen on the surface and heavily within
the zona pellucida (upper right) of this blastocyst (day 4, 4 PM)
following the concanavalin A procedure. x 42,500.
2
The reaction product penetrates between cells to the level of the
apical junctional complex in this blastocyst from day 4, 9 AM, exposed to ConA. x 42,500.
3
Peroxidase reaction product covers the surface of the trophoblast of
this zona-free blastocyst (day 5, 9 A M ) exposed to concanavalin A.
Note that the apical junctional complex is situated in an elevation,
and that the reaction product does not penetrate into this junctional
complex. The reaction product between the cells in the lower portion
of the picture apparently gained access from the interior of the
blastocyst, which it reached through one of the trophoblast cells (not
seen in the micrograph) damaged in processing. x 42,500.
4
In this control preparation exposed to ConA in the presence of
a-methyl-D-mannoside the surface is generally free of reaction product.
Day 4, 5 PM. X 27,000.
5 Ruthenium red can be seen both along the surface of the trophoblast
cell and in the zona pellucida (upper right) of this blastocyst on
day 4,4 PM. x 27,000.
38
SURFACE COATS OF MOUSE BLASTOCYST
Allen C. Enders and Sandra Schlaflre
PLATE 1
PLATE 2
EXPLANATION OF FIGURES
6
Where damage to one or more of the trophoblast cells has permitted
access of ConA to the interior of the blastocyst, the basal lamina
(BL) of the trophoblast is heavily stained with reaction product, in
addition to the usual staining of the free surface. Note the relatively
thin layer of reaction product surrounding the endodermal cell (Endo)
in the lower left. Day 5 , 9 AM. x 21,200.
7 In this micrograph of a blastocyst from day 7 of delayed implantation,
which was treated with ConA, it can be seen that the basal lamina
(BL) of the trophoblast stains more heavily than does the surface
coat. As usual, although the ConA has gained access to the cavity,
no reaction is seen in the apical junctional complex (bracket).
X 27,000.
8 Colloidal thorium dioxide is seen at the surface of this blastocyst on
day 5 , 9 AM. X 27,000.
9 A heavy coat of thorium is seen on the surface of this blastocyst on
day 7, delayed implantation. This colloidal tracer does not gain
access to the interior of the blastocyst. x 27,000.
40
SURFACE COATS OF MOUSE BLASTOCYST
Allen C. Enders and Sandra Schlafke
PLATE 2
PLATE 3
EXPLANATION OF FIGURES
10-12 ConA-peroxidase reaction product stains the surface coat of the
uterus on day 4 (9 AM), day 5 (9 AM), and day 7 of delayed implantation in figures 10,11 and 12 respectively. In no instance does
the ConA penetrate the junctional complex. Although the coat appears thinner on the day 5 uterus, the difference could well be the
result of reduced numbers of ConA binding sites within a coat of
equal thickness. x 46,500.
13 Day 5 uterus in which a-methyl-D-mannosidewas included in the
ConA and peroxidase solutions. Note the complete absence of
staining of the surface coat. x 46,500.
42
SURFACE COATS OF MOUSE BLASTOCYST
Allen C. Enders and Sandra Schlafke
PLATE 3
PLATE 4
EXPLANATION OF FIGURES
14 Extensive ruthenium red staining of the surface coat of the uterus
from day 5 (9
AM).
x 52,000.
15 Thorium dioxide binding at the surface of the uterus on day 4 (9 AM).
X 52,000.
16
44
Methylation of the uterus (day 4, 9 AM) prior to treatment with
colloidal thorium completely abolishes the binding of thorium. However, this treatment is rather harsh on the tissue. x 21,600.
SURFACE COATS O F MOUSE BLASTOCYST
Allen C. Enders and Sandra Schlafke
PLATE 4
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