estrogen-induced surface coat and enzyme changes in the...

/ . Embryol. exp. Morph. Vol. 29, 3, pp. 639-645, 1973 6 3 9

Printed in Great Britain

Estrogen-induced surface coat and enzyme changesin the implanting mouse blastocyst

By P. V. HOLMES1 AND A. D. DICKSON2

From the Division of Morphological Science, Faculty of Medicine,The University of Calgary

SUMMARYBlastocysts 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 coatof the trophoblast cells. The blastocyst surface coat was investigated using colloidal iron-Prussian blue stain. In addition, the activity of tyrosine aminotransferase was assayedhistochemically in preimplantation blastocysts. The surface and enzyme changes werecorrelated with the presence of estrogen by investigations in normal blastocysts from intactmice before and after the endogenous estrogen surge and in delayed-implantation blasto-cysts from ovariectomized mice preceding and following estrogen treatment. Colloidal ironstains the surface coat of the blastocyst after, but not before, the endogenous estrogensurge and the exogenous estrogen treatment. Tyrosine aminotransferase activity also isincreased considerably after estrogen induction.

INTRODUCTION

The preimplantation stickiness of the zona pellucida-free mouse blastocysthas been referred to previously (Boving, 1966). Whether this adhesiveness con-tributes to the attachment phase of blastocyst implantation is the concern inthis work.

In 1961, working with dissociated embryonic cells, Moscona found thatrotation of the cultures caused the cells to reaggregate. Subsequently, Moscona& Moscona (1963) were able to inhibit the adhesiveness and aggregation ofdissociated embryonic cells by blocking RNA and protein synthesis. Theysuggested that actinomycin D and puromycin may interfere with formation ofextracellular 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 themorphological characteristics of cell adhesion in embryonic cell reaggregationand 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 Univer-sity 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 aggre-gation. This possibility could apply to the attachment phase of mouse blasto-cysts, for Jones & Kemp (1969) observed, on day 6 of gestation, deposition ofa nbrinoid material containing mucoproteins, sialic acid and hyaluronic acidon the outer surface of the trophoblast cells. A similar surface mucoproteinlayer was observed and histochemically characterized on human trophoblastby Bradbury, Billington, Kirby & Williams (1969, 1970). These investigatorsdemonstrated the presence of the surface coat using colloidal-iron stain for lightand electron microscopy.

Blocking the synthesis of new RNA and protein species induced by estrogen(Dass, Mohla & Prasad, 1969) in the blastocyst could prevent the attachmentphase of implantation. Unger & Dickson (1971) have shown that actinomycin Dand cycloheximide, administered to mice at the preimplantation stage, stops boththe estrogen-induced trophoblastic giant cell transformation (Dickson, 1963,1967) and implantation from occurring.

There is a possibility that the enzyme neuraminidase may influence theadhesion of blastocysts to the uterine luminal epithelium. Gasic & Gasic (1970)administered neuraminidase intravenously to post-coitum female mice and noteda 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 anddexamethazone phosphate and were able to induce increases in tyrosineaminotransferase (TAT) activity. Correlating this work with that of Moscona& Moscona (1963), Ballard & Tomkins (1969) linked both the synthesis ofa specific cell surface adhesive factor and the increase in intracellular TATactivity with a number of administered steroid-hormone inducers. They postu-lated that tyrosine aminotransferase may be on the pathway of steroid-inducedincreases in cell adhesion.

From the foregoing, it may be proposed that the luteinizing hormone-inducedestrogen surge of pregnancy (Shelesnyak, Kraicer & Zeilmaker, 1963) may in-duce increased enzyme activity in the trophoblast cells which alters their surfacemucoproteins and that this alteration is necessary for the attachment phaseof implantation.

MATERIALS AND METHODS

Swiss Webster albino mice were used from a random-bred colony maintainedat 22 °C with lighting controlled to provide a 10 h night centred on midnight.Mouse chow from Tecklad Inc. of Monmouth, Illinois, and drinking waterwere provided ad libitum. Surgical procedures were conducted under intraperi-toneal sodium pentobarbital anesthesia and hormone injections were givensubcutaneously in the lumbar region. The mice were killed by cervical disloca-tion and blastocysts were flushed from uterine horns with Hanks's physiologicalsaline 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, namelyintact females killed between 09.00 and 10.00 h on day 4 of gestation (i.e. priorto the endogenous estrogen stimulus for implantation), intact females killedbetween 09.00 and 10.00 h on day 5 after the endogenous estrogen stimulus,females undergoing delay of implantation, and females undergoing delayof implantation to which exogenous estrogen was administered. Delay of im-plantation was achieved by post-coital bilateral ovariectomy between 09.00 and11.00 h on day 3 of gestation and administration of 1 mg of Depo Provera(medroxyprogesterone acetate suspension, Upjohn Co.) on day 3 and again onday 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 thirdgroup, both on day 8 at 09.00 h. Blastocysts undergoing delay of implantationwere collected from these mice 36 h after the day-8 injections.

Trophoblast surface coat changes were visualized in the four mouse groupsusing the colloidal iron-Prussian blue reaction, with naphthol yellow as acounterstain and controls being treated first with a-amylase for 45 min or withneuraminidase for 60 min. After formalin fixation, blastocysts were air-driedon microscope slides before staining. The stock solution of colloidal iron wasprepared by adding 12 ml of 32% (w/v) ferric chloride solution to 750 ml ofboiling, triple-distilled water. This solution was diluted with 10 vols of glacialacetic acid immediately before use, the pH being adjusted to below 1-3, if neces-sary, and was applied to the tissue for 60 min. This procedure was followed bythree 5 min rinses in 5 % acetic acid. Colloid deposits were converted to Prussianblue by a 20 min treatment with a 1:1 mixture, prepared immediately beforeuse, of 2% aqueous potassium ferrocyanide and 2% HC1. The effectiveness ofthis stain was confirmed by control staining of goblet cells in sections of smallintestine and the specificity was confirmed by carrying the procedure throughomitting the ferric chloride treatment.

Blastocysts from four groups of mice similar to those described above wereemployed for investigating tyrosine aminotransferase activity. The enzymereaction mixture was composed of 0-05 ml of 0-33 M a-ketoglutarate, 0-2 ml of0-025 M tyrosine, 0-005 ml of tautomerase (4-3-5-0 K units/ml), 0-005 ml of0-02 M pyridoxalphosphate, 0-0025 ml of a suspension of 20 mg/ml of crystallineglutamate dehydrogenase in a 50 % aqueous glycerol solution, 0-25 ml of iodo-nitrotetrazolium 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 mixture0-15 M sodium phosphate solution at pH 7-9 was added to make up 1 ml. Thereaction was inactivated by the addition of 7 % acetic acid. Blastocysts weretreated according to this technique for 30 min, formalin-fixed, washed andmounted on microscope slides as aqueous whole mounts. No counterstain wasused. Specificity of the reaction was tested by enzyme inactivation and by incu-bation without substrate. Enzyme inactivation was accomplished by heattreatment for 60 min, at 65 °C for one group of day-5 blastocysts^and at90 °C for a second group.


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OBSERVATIONS

All day-5 and delayed-implantation blastocysts lacked a zona pellucida whenflushed from the uterus. Many of the day-4 blastocysts possessed a zonapellucida. However, those with a zona did not appear to react less to thecolloid and enzyme tests than those without a zona.

Sixty-one blastocysts were collected from day-5 intact females and allexhibited colloidal iron-Prussian blue staining of the surface-coat material ofthe 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 blasto-cysts exhibited traces of stain on one side but it was impossible to determinewhether the stain traces were consistently at the abembryonic or the embryonicpole. Fifty-four blastocysts were collected from ovariectomized, estrogen-treated mice and all exhibited light-to-heavy colloidal iron staining. From theovariectomized, vehicle-treated group, 78 blastocysts were collected, most ofwhich lacked any staining, with some showing trace amounts. The 30 day-5control blastocysts untreated with ferric chloride were not stained by thecolloidal iron. The same is true of the 32 day-5 blastocysts pretreated withneuraminidase and of the 28 pretreated with a-amylase.

In the tyrosine aminotransferase experiments all blastocysts exhibited at leasttrace amounts of enzyme activity. The lowest TAT activity observed was in the17 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 affecttheir 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 TATactivity in the embryonic and trophoblast cells of the day-5 blastocysts. Moreadvanced blastocysts collected between 12.00 and 13.00 h day 5 exhibited ex-tremely high enzyme activity in some cells of the inner cell mass, as seen inFig. 4, probably endoderm cells. On comparing the two groups of ovariectom-ized mice, the 38 blastocysts from females pretreated with estrogen were observedto 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 froman intact, untreated female mouse on day 5 of gestation. Counterstained withnaphthol yellow, x 700.Fig. 2. Colloidal iron-stained blastocyst from an intact, untreated female on day 4of gestation. Note the lack of superficial uptake of colloid. Counterstained withnaphthol yellow, x 580.Fig. 3. Blastocyst from a day 4, intact female. Note the minimal amount of formazandeposition in the cells. No counterstain used, x 650.Fig. 4. Blastocyst from a day 5, intact female. Note the heavy formazan deposits inthe trophoblast and the intense staining of some cells of the inner cell mass. Nocounterstain used, x 600.


vehicle-treated females. Once again, there was very high TAT activity in theinner cell mass cells of the estrogen-stimulated blastocysts. In all cases, theformazan from the tetrazolium salt, iodonitrotetrazolium, precipitated inthe form of large, reddish-brown crystals throughout the cells.

In all of the work where blastocysts were manipulated from one solution toanother, it was found necessary to maintain the solutions at, or above,a physiological pH because of their extreme stickiness to siliconized glass andeach other in acid solutions.

DISCUSSION

The marked alteration in colloidal iron staining of the trophoblast surfacebetween days 4 and 5 of gestation in the intact mouse indicates there is probablya change in amount or functional activity of the surface glycoproteins. Sincethis surface change coincides temporally with a number of other known changesin the blastocyst, such as the trophoblastic giant cell transformation (Dickson,1963, 1969), and since an increase in blastocyst stickiness was also observed atthis time, it seems reasonable that the surface glycoproteins could be related toattachment of the trophoblast cells to the epithelial cells of the uterine lumen.The experiments with ovariectomized mice indicate that the colloidal ironstaining or trophoblast surface changes are directly, or indirectly, the result ofan estrogen stimulus.

Also at this time, between the preimplantation and implantation stages ofthe blastocyst, a considerable increase in tyrosine aminotransferase activity wasobserved in the intact mice. This coincides temporally with surface-coat andadhesiveness changes of the blastocyst and appears to be dependent on anendogenous estrogen stimulus as indicated by the work with estrogen-stimu-lated and non-stimulated delayed-implantation blastocysts. As mentionedpreviously, steroids have been shown to be responsible for increasing both theadhesiveness of cells and the intracellular activity of tyrosine aminotransferase(Ballard & Tomkins, 1969).

The substantial effect of neuraminidase on the surface coat at implantationas 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 pellu-cida of the blastocyst are biochemically different. In addition, intravenouslyadministered neuraminidase was shown by Gasic & Gasic (1970) to suppresspregnancy in mice, a result possibly due to a neuraminidase-mediated changein the blastocyst surface coat, although recently, Kunii (1971) has shown thatchorionic and serum extracts of human chorionic gonadotrophin are inacti-vated by neuraminidase.

This research was supported by the Medical Research Council of Canada.


REFERENCES

BALLARD, P. L. & TOMKINS, G. M. (1969). Hormone induced modification of the cell surface.Nature, Loud. 224, 344-345.

BOVING, B. G. (1966). Some mechanical aspects of trophoblast penetration of the uterineepithelium in the rabbit. In Egg Implantation (ed. G. E. W. Wolstenholme & M. O'Connor),pp. 72-93. London: J. and A. Churchill.

BOWMAN, P. & MCLAREN, A. (1970). The reaction of the mouse blastocyst and its zonapellucida to enzymes in vitro. J. Embryol. exp. Morph. 24, 331-334.

BRADBURY, S., BILLINGTON, W. D., KIRBY, D. R. S. & WILLIAMS, E. A. (1969). Surfacemucin of human trophoblast. Am. J. Obstet. Gynec. 104, 416-418.

BRADBURY, S., BILLINGTON, W. D., KIRBY, D. R. S. & WILLIAMS, E. A. (1970). Histochemicalcharacterization of the surface mucoprotein of normal and abnormal human trophoblast.Histochem. J. 2, 263-274.

DASS, C. M. S., MOHLA, S. & PRASAD, M. R. N. (1969). Time sequence of action of estrogenon nucleic acid and protein synthesis in the uterus and blastocyst during delayed implanta-tion in the rat. Endocrinology 85, 528-537.

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DICKSON, A. D. (1969). Cytoplasmic changes during the trophoblastic giant cell transforma-tion of blastocysts from normal and ovariectomized mice. / . Anat. 105, 371-380.

GASIC, G. J. & GASIC, T. B. (1970). Total suppression of pregnancy in mice by post-coitaladministration of neuraminidase. Proc. natn. Acad. Sci. U.S.A. 67, 793-798.

GRANNER, D. K., HAYASHI, S., THOMPSON, E. B. & TOMKINS, G. M. (1968). Stimulation oftyrosine aminotransferase synthesis by dexamethasone phosphate in cell culture. /. molec.Biol. 35, 291-301.

JONES, B. M. & KEMP, R. B. (1969). Self-isolation of the foetal trophoblast. Nature, Lond.221, 829-831.

KUNII, H. (1971). Inactivation of HCG extracted from chorionic tissue and serum byneuraminidase. TohokuJ. exp. Med. 105, 317-326.

LIN, T. P. & FLORENCE, J. (1971). Aggregation of dissociated mouse blastomeres. ExplCell Res. 63, 220-224.

MOSCONA, A. A. (1961). Rotation-mediated histogenic aggregation of dissociated cells: aquantifiable approach to cell interactions in vitro. Expl Cell Res. 22, 455-475.

MOSCONA, M. H. & MOSCONA, A. A. (1963). Inhibition of adhesiveness and aggregation ofdissociated cells by inhibitors of protein and RNA synthesis. Science, N. Y. 142, 1070-1071.

SHEFFIELD, J. B. (1970). Studies on aggregation of embryonic cells: initial cell adhesion andthe formation of intercellular junctions. / . Morph. 132, 245-264.

SHELESNYAK, M. C, KRAICER, P. F. & ZEILMAKER, G. H. (1963). Studies on the mechanismof decidualization. 1. The estrogen surge of pseudopregnancy and pro-gravidity and itsrole in the process of decidualization. Acta Endocr. 42, 225-232.

THOMPSON, E. B., TOMKINS, G. M. & CURRAN, J. F. (1966). Induction of tyrosine a-keto-glutarate transaminase by steroid hormones in a newly established tissue culture cell line.Proc. natn. Acad. Sci. U.S.A. 56, 296-303.

UNGER, B. & DICKSON, A. D. (1971). Effect of cycloheximide and actinomycin D on themouse blastocyst undergoing the giant cell transformation. / . Anat. 108, 519-525.

{Received 18 September 1972, revised 25 November 1972)

estrogen-induced surface coat and enzyme changes in the...

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