/ . Embryol. exp. Morph. Vol. 29, 3, pp. 639-645, 1973
Printed in Great Britain
639
Estrogen-induced surface coat and enzyme changes
in the implanting mouse blastocyst
By P. V. HOLMES 1 AND A. D. DICKSON 2
From the Division of Morphological Science, Faculty of Medicine,
The University of Calgary
SUMMARY
Blastocysts from estrogen-stimulated mice tend to be very sticky, a characteristic which
can be correlated with estrogen-induced enzyme activation and changes in the surface coat
of the trophoblast cells. The blastocyst surface coat was investigated using colloidal ironPrussian blue stain. In addition, the activity of tyrosine aminotransferase was assayed
histochemically in preimplantation blastocysts. The surface and enzyme changes were
correlated with the presence of estrogen by investigations in normal blastocysts from intact
mice before and after the endogenous estrogen surge and in delayed-implantation blastocysts from ovariectomized mice preceding and following estrogen treatment. Colloidal iron
stains the surface coat of the blastocyst after, but not before, the endogenous estrogen
surge and the exogenous estrogen treatment. Tyrosine aminotransferase activity also is
increased considerably after estrogen induction.
INTRODUCTION
The preimplantation stickiness of the zona pellucida-free mouse blastocyst
has been referred to previously (Boving, 1966). Whether this adhesiveness contributes to the attachment phase of blastocyst implantation is the concern in
this work.
In 1961, working with dissociated embryonic cells, Moscona found that
rotation of the cultures caused the cells to reaggregate. Subsequently, Moscona
& Moscona (1963) were able to inhibit the adhesiveness and aggregation of
dissociated embryonic cells by blocking RNA and protein synthesis. They
suggested that actinomycin D and puromycin may interfere with formation of
extracellular materials having specific cell-binding roles. Lin & Florence (1971)
dissociated the cells of four- to eight-cell mouse ova and observed reaggregation,
some aggregates developing to the blastocyst stage. Sheffield (1970) studied the
morphological characteristics of cell adhesion in embryonic cell reaggregation
and postulated that an adhesive material may be released on the cell surface to
1
Author's address: Institute of Human Anatomy, University of Uppsala, Uppsala,
Sweden.
2
Author's address: Division of Morphological Science, Faculty of Medicine, The University of Calgary, Calgary 44, Alberta, Canada.
640
P. V. HOLMES AND A. D. DICKSON
provide a means of cell-cell recognition and attachment for histogenetic aggregation. This possibility could apply to the attachment phase of mouse blastocysts, for Jones & Kemp (1969) observed, on day 6 of gestation, deposition of
a nbrinoid material containing mucoproteins, sialic acid and hyaluronic acid
on the outer surface of the trophoblast cells. A similar surface mucoprotein
layer was observed and histochemically characterized on human trophoblast
by Bradbury, Billington, Kirby & Williams (1969, 1970). These investigators
demonstrated the presence of the surface coat using colloidal-iron stain for light
and electron microscopy.
Blocking the synthesis of new RNA and protein species induced by estrogen
(Dass, Mohla & Prasad, 1969) in the blastocyst could prevent the attachment
phase of implantation. Unger & Dickson (1971) have shown that actinomycin D
and cycloheximide, administered to mice at the preimplantation stage, stops both
the estrogen-induced trophoblastic giant cell transformation (Dickson, 1963,
1967) and implantation from occurring.
There is a possibility that the enzyme neuraminidase may influence the
adhesion of blastocysts to the uterine luminal epithelium. Gasic & Gasic (1970)
administered neuraminidase intravenously to post-coitum female mice and noted
a total suppression of pregnancy, an effect reversible by progesterone treatment.
Thompson, Tomkins & Curran (1966) and Granner, Hayashi, Thompson
& Tomkins (1968) treated cell cultures, respectively, with glucocorticoids and
dexamethazone phosphate and were able to induce increases in tyrosine
aminotransferase (TAT) activity. Correlating this work with that of Moscona
& Moscona (1963), Ballard & Tomkins (1969) linked both the synthesis of
a specific cell surface adhesive factor and the increase in intracellular TAT
activity with a number of administered steroid-hormone inducers. They postulated that tyrosine aminotransferase may be on the pathway of steroid-induced
increases in cell adhesion.
From the foregoing, it may be proposed that the luteinizing hormone-induced
estrogen surge of pregnancy (Shelesnyak, Kraicer & Zeilmaker, 1963) may induce increased enzyme activity in the trophoblast cells which alters their surface
mucoproteins and that this alteration is necessary for the attachment phase
of implantation.
MATERIALS AND METHODS
Swiss Webster albino mice were used from a random-bred colony maintained
at 22 °C with lighting controlled to provide a 10 h night centred on midnight.
Mouse chow from Tecklad Inc. of Monmouth, Illinois, and drinking water
were provided ad libitum. Surgical procedures were conducted under intraperitoneal sodium pentobarbital anesthesia and hormone injections were given
subcutaneously in the lumbar region. The mice were killed by cervical dislocation and blastocysts were flushed from uterine horns with Hanks's physiological
saline and fixed for 24 h in 10% buffered neutral formalin.
Estrogen-induced changes in the mouse blastocyst
641
Blastocysts were collected from four experimental groups of mice, namely
intact females killed between 09.00 and 10.00 h on day 4 of gestation (i.e. prior
to the endogenous estrogen stimulus for implantation), intact females killed
between 09.00 and 10.00 h on day 5 after the endogenous estrogen stimulus,
females undergoing delay of implantation, and females undergoing delay
of implantation to which exogenous estrogen was administered. Delay of implantation was achieved by post-coital bilateral ovariectomy between 09.00 and
11.00 h on day 3 of gestation and administration of 1 mg of Depo Provera
(medroxyprogesterone acetate suspension, Upjohn Co.) on day 3 and again on
day 8 at 09.00 h. Exogenous estrogen (estradiol benzoate, 0-05 jug in corn oil)
was administered to the fourth experimental group and vehicle only to the third
group, both on day 8 at 09.00 h. Blastocysts undergoing delay of implantation
were collected from these mice 36 h after the day-8 injections.
Trophoblast surface coat changes were visualized in the four mouse groups
using the colloidal iron-Prussian blue reaction, with naphthol yellow as a
counterstain and controls being treated first with a-amylase for 45 min or with
neuraminidase for 60 min. After formalin fixation, blastocysts were air-dried
on microscope slides before staining. The stock solution of colloidal iron was
prepared by adding 12 ml of 32% (w/v) ferric chloride solution to 750 ml of
boiling, triple-distilled water. This solution was diluted with 10 vols of glacial
acetic acid immediately before use, the pH being adjusted to below 1-3, if necessary, and was applied to the tissue for 60 min. This procedure was followed by
three 5 min rinses in 5 % acetic acid. Colloid deposits were converted to Prussian
blue by a 20 min treatment with a 1:1 mixture, prepared immediately before
use, of 2 % aqueous potassium ferrocyanide and 2 % HC1. The effectiveness of
this stain was confirmed by control staining of goblet cells in sections of small
intestine and the specificity was confirmed by carrying the procedure through
omitting the ferric chloride treatment.
Blastocysts from four groups of mice similar to those described above were
employed for investigating tyrosine aminotransferase activity. The enzyme
reaction mixture was composed of 0-05 ml of 0-33 M a-ketoglutarate, 0-2 ml of
0-025 M tyrosine, 0-005 ml of tautomerase (4-3-5-0 K units/ml), 0-005 ml of
0-02 M pyridoxalphosphate, 0-0025 ml of a suspension of 20 mg/ml of crystalline
glutamate dehydrogenase in a 50 % aqueous glycerol solution, 0-25 ml of iodonitrotetrazolium at 32 mg/ml, 0-1 ml of diphosphopyridine nucleotide at 10 mg/
ml, and 005ml of 0-4mg/ml of phenazine methosulphate. To this mixture
0-15 M sodium phosphate solution at pH 7-9 was added to make up 1 ml. The
reaction was inactivated by the addition of 7 % acetic acid. Blastocysts were
treated according to this technique for 30 min, formalin-fixed, washed and
mounted on microscope slides as aqueous whole mounts. No counterstain was
used. Specificity of the reaction was tested by enzyme inactivation and by incubation without substrate. Enzyme inactivation was accomplished by heat
treatment for 60 min, at 65 °C for one group of day-5 blastocysts^and at
90 °C for a second group.
642
P. V. HOLMES AND A. D. DICKSON
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Estrogen-induced changes in the mouse blastocyst
643
OBSERVATIONS
All day-5 and delayed-implantation blastocysts lacked a zona pellucida when
flushed from the uterus. Many of the day-4 blastocysts possessed a zona
pellucida. However, those with a zona did not appear to react less to the
colloid and enzyme tests than those without a zona.
Sixty-one blastocysts were collected from day-5 intact females and all
exhibited colloidal iron-Prussian blue staining of the surface-coat material of
the trophoblast cells (Fig. 1). Of the 87 collected from day-4 intact females,
almost all lacked any surface-coat staining (Fig. 2). A very few of these blastocysts exhibited traces of stain on one side but it was impossible to determine
whether the stain traces were consistently at the abembryonic or the embryonic
pole. Fifty-four blastocysts were collected from ovariectomized, estrogentreated mice and all exhibited light-to-heavy colloidal iron staining. From the
ovariectomized, vehicle-treated group, 78 blastocysts were collected, most of
which lacked any staining, with some showing trace amounts. The 30 day-5
control blastocysts untreated with ferric chloride were not stained by the
colloidal iron. The same is true of the 32 day-5 blastocysts pretreated with
neuraminidase and of the 28 pretreated with a-amylase.
In the tyrosine aminotransferase experiments all blastocysts exhibited at least
trace amounts of enzyme activity. The lowest TAT activity observed was in the
17 substrate-control blastocysts and the 29 90 °C heat-control blastocysts.
Treatment of 29 day-5 blastocysts for 60 min at 65 °C did not appear to affect
their TAT activity. A comparison between 46 day-4 (Fig. 3) and 48 day-5
(Fig. 4) blastocysts from intact mice demonstrates a marked increase in TAT
activity in the embryonic and trophoblast cells of the day-5 blastocysts. More
advanced blastocysts collected between 12.00 and 13.00 h day 5 exhibited extremely high enzyme activity in some cells of the inner cell mass, as seen in
Fig. 4, probably endoderm cells. On comparing the two groups of ovariectomized mice, the 38 blastocysts from females pretreated with estrogen were observed
to have very much higher formazan deposition than the 48 blastocysts from the
FIGURES 1-4
Fig. 1. Colloidal iron-Prussian blue staining of the surface coat of a blastocyst from
an intact, untreated female mouse on day 5 of gestation. Counterstained with
naphthol yellow, x 700.
Fig. 2. Colloidal iron-stained blastocyst from an intact, untreated female on day 4
of gestation. Note the lack of superficial uptake of colloid. Counterstained with
naphthol yellow, x 580.
Fig. 3. Blastocyst from a day 4, intact female. Note the minimal amount of formazan
deposition in the cells. No counterstain used, x 650.
Fig. 4. Blastocyst from a day 5, intact female. Note the heavy formazan deposits in
the trophoblast and the intense staining of some cells of the inner cell mass. No
counterstain used, x 600.
644
P. V. HOLMES AND A. D. DICKSON
vehicle-treated females. Once again, there was very high TAT activity in the
inner cell mass cells of the estrogen-stimulated blastocysts. In all cases, the
formazan from the tetrazolium salt, iodonitrotetrazolium, precipitated in
the form of large, reddish-brown crystals throughout the cells.
In all of the work where blastocysts were manipulated from one solution to
another, it was found necessary to maintain the solutions at, or above,
a physiological pH because of their extreme stickiness to siliconized glass and
each other in acid solutions.
DISCUSSION
The marked alteration in colloidal iron staining of the trophoblast surface
between days 4 and 5 of gestation in the intact mouse indicates there is probably
a change in amount or functional activity of the surface glycoproteins. Since
this surface change coincides temporally with a number of other known changes
in the blastocyst, such as the trophoblastic giant cell transformation (Dickson,
1963, 1969), and since an increase in blastocyst stickiness was also observed at
this time, it seems reasonable that the surface glycoproteins could be related to
attachment of the trophoblast cells to the epithelial cells of the uterine lumen.
The experiments with ovariectomized mice indicate that the colloidal iron
staining or trophoblast surface changes are directly, or indirectly, the result of
an estrogen stimulus.
Also at this time, between the preimplantation and implantation stages of
the blastocyst, a considerable increase in tyrosine aminotransferase activity was
observed in the intact mice. This coincides temporally with surface-coat and
adhesiveness changes of the blastocyst and appears to be dependent on an
endogenous estrogen stimulus as indicated by the work with estrogen-stimulated and non-stimulated delayed-implantation blastocysts. As mentioned
previously, steroids have been shown to be responsible for increasing both the
adhesiveness of cells and the intracellular activity of tyrosine aminotransferase
(Ballard & Tomkins, 1969).
The substantial effect of neuraminidase on the surface coat at implantation
as seen in this work compared with the lack of its effect on the zona pellucida
(Bowman & McLaren, 1970) indicates that the surface coat and the zona pellucida of the blastocyst are biochemically different. In addition, intravenously
administered neuraminidase was shown by Gasic & Gasic (1970) to suppress
pregnancy in mice, a result possibly due to a neuraminidase-mediated change
in the blastocyst surface coat, although recently, Kunii (1971) has shown that
chorionic and serum extracts of human chorionic gonadotrophin are inactivated by neuraminidase.
This research was supported by the Medical Research Council of Canada.
Estrogen-induced changes in the mouse blastocyst
645
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{Received 18 September 1972, revised 25 November 1972)